Compositions And Methods For In Vivo Imaging

ABSTRACT

This document relates to compounds useful for targeting PARP1. Also provided herein are methods for using such compounds to detect and image cancer cells.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.16/147,370, filed Sep. 28, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/581,386, filed on Apr. 28, 2017, now U.S. Pat.No. 10,117,954, which is a continuation of U.S. patent application Ser.No. 13/988,790, filed with a 371 filing date of Aug. 2, 2013, now U.S.Pat. No. 9,649,394, which is a U.S. National Phase Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2011/061856,filed on Nov. 22, 2011, which claims priority under 35 U.S.C. § 119(e)to U.S. Patent Application Ser. Nos. 61/419,929, filed on Dec. 6, 2010,and 61/416,035, filed on Nov. 22, 2010, all of which are incorporatedherein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with government support under Grant Nos.P50 CA86 355 and NIBI-RO1 EB-010 011 awarded by the National CancerInstitute; and Grant No. NIGMS T32 GM008313 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

TECHNICAL FIELD

This document relates to detectable compounds that bind PARP1. Alsoprovided herein are methods for using such compounds to detect and imagecells containing PARP1, for example, cancer cells.

BACKGROUND

Poly(ADP-ribose) polymerase 1 (PARP1) is an important cellular proteinthat senses DNA damage and initiates the base excision repair pathway.DNA damage (e.g., strand breaks) occurs during each cell cycle and mustbe repaired for a cell to survive. In cells undergoing rapid divisionand proliferation, such as cancer cells, levels of PARP1 aresignificantly increased. This is particularly true in cells lackingother DNA repair enzymes, such as cells lacking function BRCA1 andBRCA2.

SUMMARY

Described herein are detectable compounds that bind PARP1. Also providedherein are methods and materials for using such compounds to image cellscontaining PARP1, for example, cancer cells. In some cases, such cellsoverexpress PARP1. For example, a compound of Formula (1) can be used todetect or image a cancer cell in a subject through noninvasive imagingof the subject.

The methods and compositions provided herein provide several advantages.For example, one problem in the assessment of therapeutic efficacy ofcancer therapies has been the inability to image PARP1 noninvasively atthe whole-body level and to quantitate therapeutic inhibition. With thepresent technology it is possible to separate subjects into appropriatetreatment groups and to detect emerging resistance. Given the emergingimportance of positron emission tomography (PET) imaging in preclinicaland clinical settings, and evidence that fluorescently labeled PARP1inhibitors can be used for cellular imaging, the compounds providedherein function as detectable probes for whole-body PARP1 imaging. Insome embodiments, a compound provided herein is useful for measuringinhibition of PARP1 by emerging therapeutic PARP1 inhibitors.

In addition to the advantage described above, one way to identify usefulimaging agents is to empirically test a series of different agents invivo. This approach, however, places considerable demands on thelabeling of each compound. To overcome these lengthy sequentialoptimization procedures for each individual compound, provided herein isone such approach based on the combination of [4+2] inverse electrondemand Diels-Alder cycloadditions; its utility is shown by thedevelopment of the novel PARP1 imaging agents described herein.

This disclosure provides a compound of Formula (I):

P-L_(n)-T_(m)-D

or a pharmaceutically acceptable salt form thereof,wherein:P is a PARP1 inhibitor;L is a linker;

T is:

D is a detectable agent;m is 0 or 1; andn is 0 or 1.

In some embodiments, P is selected from the group consisting of:benzamide, quinolone, dihydroisoquinolinone, isoquinolinone,isoquinolone, benzopyrone, cyclic benzamide, benzimidazole, indole,isoindolinone, nicotinamide, 3-AB, phthalazinone, and quinazolinone. Forexample, P can be selected from the group consisting of: AZD2281(olaparib), AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib),BSI101, DR2313, FR 247304, GPI15427, GPI16539, MK4827, NU1025, NU1064,NU1085, PD128763, PARP Inhibitor II (INH2BP), PARP Inhibitor III (DPQ),PARP Inhibitor VIII (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A. Insome embodiments, P is AZD2281.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II):

or a pharmaceutically acceptable salt form thereof,wherein:L is a linker;

T is:

D is a detectable agent;m is 0 or 1; andn is 0 or 1.

In some embodiments, D comprises one or more of organic small molecules,inorganic compounds, nanoparticles, enzymes or enzyme substrates,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, and contrast agents. For example, D can comprisea radioactive material, a fluorescent material, or a mixture thereof.

Non-limiting examples of a compound of Formula (I) include:

wherein R is Texas Red, a cyanine dye, Alexafluor-680, a BODIPY dye, axanthene derivatives, a naphthalene dye, a courmarin derivative, anoxadiazole derivative, a pyrene derivative, an oxazine derivative, anacridine derivative, an arylmethine derivative, and a tetrapyrrolederivative.

In some embodiments, the compound of Formula (1) is selected from thegroup consisting of:

In some embodiments, a compound of Formula (1) is:

For example, the compound of Formula (1) can be:

Also provided herein is a pharmaceutical composition comprising acompound of Formula (I), or a pharmaceutically acceptable salt formthereof.

The compounds provided herein are useful for detecting a cancer in asubject. In some embodiments, the method comprises: administering to asubject an effective amount of a compound of Formula (I), or apharmaceutically acceptable salt form thereof, and detecting thedetectable agent in the subject. In some embodiments, the compound ofFormula (1) is administered before and after surgical removal of thecancer.

In some embodiments, detecting the detectable agent comprises using oneor more of histochemistry, fluorescence detection, chemiluminescencedetection, bioluminescence detection, magnetic resonance imaging,nuclear magnetic resonance imaging, positron emission tomography,single-photon emission computed tomography, X-ray imaging, X-raycomputed tomography, ultrasound imaging, or photoacoustic imaging.

In some embodiments, the cancer is selected from the group consistingof: pancreatic cancer, ovarian cancer, and breast cancer.

Also provided herein is a method for imaging a cancer cell, the methodcomprising: contacting a cancer cell with an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt formthereof, and imaging the cell.

Further provided herein is a method for monitoring the cancer treatmentof a patient, the method comprising: (a) administering to the patient,prior to a treatment, an effective amount of a compound of Formula (I),or a pharmaceutically acceptable salt form thereof, and imaging thepatient; (b) administering to the patient, at a point followingtreatment, an effective amount the compound of Formula (I) and imagingthe patient; and (c) comparing the image collected in step (a) with theimage collected in step (b) to monitor the treatment. In someembodiments, the treatment comprises administration of an anti-canceragent. In some embodiments, the method further comprises: administeringto the patient an effective amount of the compound of Formula (I) duringtreatment and imaging the patient.

Definitions

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” ismeant to account for variations due to experimental error. Allmeasurements reported herein are understood to be modified by the term“about”, whether or not the term is explicitly used, unless explicitlystated otherwise. As used herein, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

A “subject,” as used herein, includes both humans and other animals,particularly mammals. Thus, the methods are applicable to both humantherapy and veterinary applications. In some embodiments, the subject isa mammal, for example, a primate. In some embodiments, the subject is ahuman.

An “effective” amount of a compound provided herein is typically onewhich is sufficient to allow detection of the compound (e.g., detectionof a cancer cell) and may vary according to the detection methodutilized and the detection limit of the compound.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, constitutional isomers, and tautomersof the structures depicted. Compounds herein identified by name orstructure as one particular tautomeric form are intended to includeother tautomeric forms unless otherwise specified.

In some embodiments, a compound provided herein, or salt thereof, issubstantially isolated. By “substantially isolated” is meant that thecompound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compound providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compound provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is an HPLC trace of AZD2281-TCO (7); Texas Red-tetrazine (8; oneisolated stereoisomer shown); and AZD2281-Texas Red (9; crude reactionmixture). FIG. 1B shows an LC-MS spectrum of AD2281-Texas Red (9; LC-MSof major product peak shown).

FIG. 2A illustrates the reaction of AZD2281-TCO (7) and Texas Red-Tz (8)in MDA-MB436 cells. FIG. 2A shows AZD2281-TCO reacted with Texas Red-Tz;speckles outside the cells, presumably resulting from precipitated TexasRed-Tz (8), were removed; FIG. 2B shows anti-PARP1 monoclonal antibodystaining; FIG. 2C is a composite overlay on phase contrast; and FIG. 2Dillustrates the ratio of nuclear/cytoplasmic signal forAZD2281-TCO/Texas Red-Tz in the absence (not blocked: circle) andpresence (blocked: square) of blocking reagent AZD2281. Scale Bar: 20mm.

FIG. 3 shows IC₅₀ curves for the bifunctional 1(2H)-phthalazinone-basedtargeted probes.

FIG. 4 is a line graph representing IC₅₀ curves for PARP1 inhibitors (9)(circle), (10^(19F)) (square), (17^(19F)) (inverted triangle) andtrans-cyclooctene (6^(19F)) (triangle).

FIG. 5A is a schematic illustrating the synthesis of ¹⁸F-AZD2281 (6);¹⁸F-labeled TCO 3 and AZD2281-Tz (5) were combined and incubated for 3minutes; magnetic TCO-scavenger resin was added, incubated for 5minutes, and removed; purified ¹⁸F-AZD2281 was reconstituted and broughtinto an injectable volume. FIG. 5B illustrates the synthesis of themagnetic TCO-scavenger resin from amine-decorated beads andNETS-activated TCO (1). FIG. 5C illustrates the synthesis of ¹⁸F-labeledTCO (3). FIG. 5D provides the structure of AZD2281 (4). FIG. 5E showsthe synthesis and structure of ¹⁸F-AZD2281 ((6); only one isomer shown).FIG. 5F illustrates the radioactivity and absorption traces of the¹⁸F-AZD2281 reaction mixture before and after purification with themagnetic TCO-scavenger resin.

FIG. 6 shows the results of competitive in vitro inhibition assays with¹⁸F-AZD2281. Cells containing either a high (MDA-MB-436) or a low amountof PARP1 (MDA-MB-231) were treated with 5 mCi of ¹⁸F-AZD2281 in thepresence of different concentrations of AZD2281 (10 μM-0.01 nM). Afterthe cells were washed, cell-associated activity was determined bymeasurement of the g-radiation. For the control measurement, nounlabeled AZD2281 was added.

FIGS. 7A-C illustrate an in vivo evaluation of ¹⁸F-AZD2281. FIG. 7Ashows combined PET/CT scans of a nontumor-bearing mouse injected with¹⁸F-AZD2281 recorded 10, 30, and 50 minutes after injection. FIG. 7Bshows the three-dimensional reconstruction of a tumor-bearing animalinjected with ¹⁸F-AZD2281 with and without pre-injection of AZD2281(bladder segmented out for clarity). FIG. 7C illustrates quantificationof uptake through the tumor in hind legs with and withoutintraperitoneal pre-injection of unlabeled AZD2281 (SUV: standardizeduptake value).

FIG. 8A illustrates the ability of FDG and ¹⁸F-AZD2281 to distinguishbetween muscle and tumor cells. FIG. 8B shows the relative uptake of¹⁸F-AZD2281 activity of different ovarian and pancreatic tumors (A2780and SKOV3=ovarian, PANC1 and PACA2=pancreatic).

FIG. 9A shows that AZD2281-BODIPY FL (green) localizes with the nucleus(as confirmed with the red signal, Chromatin (essentially a DNA stain)).FIGS. 9B and 9C show the amount of fluorescence present in cells usingAZD2281 and a PARP1/PARP2 antibody.

FIG. 10 illustrates the life cell imaging of three different AZD2281fluorophores.

FIG. 11 shows line graphs illustrating the life cell imaging of threedifferent AZD2281 fluorophores.

FIG. 12 shows the behavior of AZD281-BODIPY FL in vivo following ivadministration of the compound into a mouse.

FIG. 13A is a bar graph illustrating the uptake of AZD2281-BODIPY FL ona global level; FIG. 13B shows the uptake of AZD2281-BODIPY FL on acellular level.

FIG. 14 illustrates various ¹⁸F-AZD2281-BODIPY compounds for in vivoimaging.

FIGS. 15A and 15B are schemes illustrating the preparation of¹⁸F-AZD2281-BODIPY FL compounds.

DETAILED DESCRIPTION

Described herein are detectable compounds that bind PARP1. Also providedherein are methods and materials for using such compounds to image cellscontaining PARP1, for example, cancer cells. In some cases, such cellsoverexpress PARP1. For example, a compound of Formula (1) or (2) can beused to detect or image a cancer cell, e.g., in a living subject, e.g.,through noninvasive imaging of the subject.

Compounds

Provided herein are compounds of Formula (1):

P-L_(n)-T_(m)-D

or a pharmaceutically acceptable salt form thereof,wherein:P is a PARP1 inhibitor;L is a linker;

T is:

D is a detectable agent;m is 0 or 1; andn is 0 or 1.

A PARP1 inhibitor can include any compound which inhibits or reduces theactivation of PARP1. For example, PARP1 activation in response to DNAbreaks and/or involved in cell death. PARP1 inhibitors include compoundsfrom various chemical classes including benzamides, quinolones,dihydroisoquinolinones, isoquinolinones, isoquinolones, benzopyrones,cyclic benzamides, benzimidazoles, indoles, isoindolinones,nicotinamides, 3-AB, phthalazinones, quinazolinones, phenanthridinones,and zinc-fingers. In some embodiments, a PARP1 inhibitor is selectedfrom the group consisting of: AZD2281 (olaparib), AG014699 (rucaparib),ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304,GPI15427, GPI16539, MK4827, NU1025, NU1064, NU1085, PD128763, PARPInhibitor II (INH2BP), PARP Inhibitor III (DPQ), PARP Inhibitor VIII(PJ34), PARP Inhibitor IX (EB-47), and TIQ-A. For example, a PARP1inhibitor can be AZD2281.

In some embodiments, a compound of Formula (1) is a compound of Formula(2):

or a pharmaceutically acceptable salt form thereof,wherein:L is a linker;

T is:

D is a detectable agent;m is 0 or 1; andn is 0 or 1.

Examples of detectable agents include various organic small molecules,inorganic compounds, nanoparticles, enzymes or enzyme substrates,fluorescent materials, luminescent materials, bioluminescent materials,chemiluminescent materials, magnetic materials, radioactive materials,and contrast agents. In some embodiments, the detectable agent is aradioactive material or a fluorescent material. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitablefluorescent materials include boron-dipyrromethene (BODIPY®),4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid(BODIPY® FL),6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoicacid, succinimidyl ester (BODIPY® TRM-X), Oregon Green 88,6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid, succinimidyl ester (BODIPY® 650/665-X),7-N,N-diethylaminocoumarin, sulforhodamine 101 acid chloride (TexasRed), VIVOTAG 680 (an amine-reactive near-infra-red fluorochrome, fromVisEn Medical), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin; an example of a luminescent material includes luminol;examples of bioluminescent materials include luciferase, luciferin, andaequorin, and examples of suitable radioactive material include ¹¹C,¹³N, ¹⁵O, ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ⁸⁶Y, ⁸⁹Zr, ¹¹¹In, ¹²³I, ¹²⁴I,¹³³Xe, ²⁰¹Tl, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, ⁶⁴Cu, ^(99m)Tc (e.g., aspertechnetate (technetate(VII), TcO₄ ⁻) either directly or indirectly,¹²³I, or other radioisotope detectable by direct counting ofradioemmission or by scintillation counting. In some embodiments, theradioactive material is complexed by a suitable ligand modality. Suchligands are well known in the art. For example, a ligand can includeDTPA, DTPE, DOTA, NOTA, DO3A, DDPE, and DOTAGA. In some embodiments, aradioisotope is conjugated to the compound directly or indirectly via alinker (e.g., an alkyl or ether linker). In addition, contrast agents,e.g., contrast agents for MM or NMR, for X-ray CT, Raman imaging,optical coherence tomography, absorption imaging, ultrasound imaging, orthermal imaging can be used. Exemplary contrast agents include gold(e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides(e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons can also beused.

In some embodiments, the detectable agent is a non-detectable pre-cursorthat becomes detectable upon activation. Examples include fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE (VisEn Medical))

When the compounds are enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, theenzymatic label is detected by determination of conversion of anappropriate substrate to product.

In some embodiments, the detectable agent is selected from the groupconsisting of: ¹⁸F, Texas Red, BODIPY FL, BODIPY 578, and BODIPY 650.

In vitro assays in which these compositions can be used include enzymelinked immunosorbent assays (ELISAs), immunoprecipitations,immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA),and Western blot analysis.

In some embodiments, a detectable agent comprises one or more of thedetectable agents described above. For example, a fluorescent detectableagent can have one or more atoms replaced with one or moreradionuclides. In some embodiments, a fluorophore comprising one or morefluorine atoms can have one or more of those fluorine atomsradioactively labeled with ¹⁸F. Non-limiting examples include ¹⁸F-BODIPYFL, ¹⁸F-BODIPY 578, and ¹⁸F-BODIPY 650. See, for example, FIG. 14.

The term “linker” as used herein refers to a group of atoms, e.g., 0-500atoms, and can be comprised of the atoms or groups of atoms such as, butnot limited to, hydrogen and carbon, e.g., methylene (—CH₂—), amino,amido, alkylamino, oxygen, polyethylene glycol (PEG), peptoid, sulfur,sulfoxide, sulfonyl, carbonyl, and imine. The linker can also comprisepart of a saturated, unsaturated or aromatic ring, including polycyclicand heteroaromatic rings wherein the heteroaromatic ring is an arylgroup containing from one to four heteroatoms, N, O or S. Specificexamples include, but are not limited to, saturated alkanes, alkylethers, alkyl diamines, and alkyl chains having one or more peptidebonds. In some embodiments, the linker is linked to the PARP1 inhibitor,the T moiety, or a detectable agent through a peptide bond. The linkermust not interfere with the imaging methods or with the bioorthogonalconjugation reactions described herein.

Non-limiting examples of linkers provided herein include:

-   wherein each p is independently an integer from 1 to 20 (e.g., 1 to    15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 2 to 20, 4 to 20, 5 to 20, 8    to 20, 12 to 20, 15 to 20, 2 to 8, and 1 to 6); and-   r is an integer from 1 to 20 (e.g., 1 to 15, 1 to 12, 1 to 10, 1 to    8, 1 to 5, 2 to 20, 4 to 20, 5 to 20, 8 to 20, 12 to 20, 15 to 20, 2    to 8, and 1 to 6).

In some embodiments, p is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5,or 6). In some embodiments, r is an integer from 1 to 3 (e.g., 1, 2, or3). In some embodiments, a linker is selected from the group consistingof:

Non-limiting examples of a compound of Formula (1) or Formula (2)include:

wherein R is Texas Red, cyanine dyes (e.g., VivoTag-680, Cy5),Alexafluor-680, BODIPY dyes (e.g., BODIPY-FL), Xanthene derivatives(e.g., Fluorescein), Naphthalene dyes (e.g., Dansyl), Courmarinderivatives, oxadiazole derivatives, pyrene derivatives, oxazinederivatives, acridine derivatives, arylmethine derivatives, tetrapyrrolederivatives. In some embodiments, R is Texas Red.

For example, a compound of Formula (1) or Formula (2) can include:

In some embodiments, the compound of Formula (1) or Formula (2) is:

Compounds provided herein, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

For example, AZD2281-Texas Red can be prepared, in part, as shown in thefollowing scheme:

Briefly, for the synthesis of the bioorthogonally reactive derivativesAZD2281-NOB (6) and AZD2281-TCO (7), compound (2) was first reacted withglutaric acid anhydride to produce the glutaric acid-modified4-(5-oxopentanamide)piperizine (3). Subsequently, an ethylene diaminespacer was attached to precursor (3), yielding the amine-functionalizedAZD2281 derivative (4). Norbornene-functionalized AZD2281-NOB (6) wasobtained by amide-bond formation with 5-norbornene-2-carboxylic acid inthe presence of polymer supported dicyclohexylcarbodiimide (DCC) beads.In the case of AZD2281-trans-cyclooctene (AZD2281-TCO; 7), precursor (3)was reacted with (E)-cyclooct-4-enyl 2,5-dioxopyrrolidin-1-yl carbonate(5) in the presence of triethylamine. Cycloaddition of both AZD2281-TCO(7) and Texas Red-Tz (8) was detected by mixing the two compounds (0.3mm), agitating for several minutes, and analyzing the products byHPLC-MS.

As another example, preparation of AZD2281-¹⁸F can be as follows:

Briefly, (Z)-2-(Cyclooct-4-enyloxy)acetic acid (2) was prepared in 63%yield over two steps from commercially available9-oxabicyclo[6.1.0]non-4-ene. Carboxylic acid (2) was converted to(E)-2-(cyclooct-4-enyloxy)ethanol (4) first by lithium aluminum hydride(LiAlH₄) reduction to give (Z)-2-(cyclooct-4-enyloxy)ethanol (3) in 78%yield, followed by photochemical cis/trans isomerization and isolationof the (E)-isomers by the previously described cycle/trap method (M.Royzen et al., J. Am. Chem. Soc. 2008, 130, 3760-3761). The major(E)-cyclooctyl stereoisomer was isolated by column chromatography andconverted to the corresponding tosylate (5) in 84% yield.(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (6^(18F)) was prepared in 91% yieldby the treatment of (5) with tetrabutylammonium fluoride (TBAF) in THF.All previously unknown compounds were fully characterized by ¹H, ¹³C,and ¹⁸F NMR. As a precursor for the chemoselective reactive PARP1inhibitor AZD2281-Tz (9), 4-[[4-fluoro-3-(4-(5-oxopentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (7) wasgenerated as described herein (See Example 1). This precursor wasreacted with (8) in the presence of polymer-supportedN,N′-dicyclohexylcarbodiimide (DCC) beads to yield (9) as a pink solid.Cycloadduct (10^(18F)) was prepared by the addition of dimethylsulfoxide (DMSO) solutions of (9) and (6^(18F)) at room temperature andsubsequent high pressure liquid chromatography (HPLC) purification.

In some embodiments, wherein a compound of Formula (1) comprises a Tmoiety, preparation of the compound can include the use of bioorthogonalconjugation reactions such as the inverse electron demand Diels-Alderreaction. For example, in a compound of Formula (1) or Formula (2), Tcan be the reaction product of an inverse electron demand Diels-Alderreaction. This reaction employs a diene and a dienophile. The reactionof a diene (e.g., a substituted tetrazine) with a dienophile (e.g., analkene or alkyne), produces an unstable cycloadduct that subsequentlyundergoes a retro-Diels-Alder cycloaddition reaction to producedinitrogen as a byproduct and the desired dihydropyrazine (afterreaction with an alkene) or pyrazine (after reaction with an alkyne)products (shown below). See e.g., Sauer et al., Chem Ber 998: 1435-1445,1965, which is incorporated by reference in its entirety. Thedihydropyrazine product may undergo an additional oxidation step togenerate the corresponding pyrazine.

A variety of tetrazines and dienophiles including cyclic and linearalkenes or alkynes have been studied in this reaction. Selection of theappropriate reaction partners allows for tuning of the coupling rate byseveral orders of magnitude. (Balcar J et al., 1983, Tet Lett24:1481-1484; Thalhammer F et al., 1990, Tet Lett 47:6851-6854). Seealso US 2006/0269942, WO 2007/144200, US 2008/0181847 and US2011/0268654. Bioconjugation methods using inverse electron demandDiels-Alder cycloadditions between tetrazines and highly straineddienophiles such as norbornene and trans-cyclooctene are known in theliterature, however the tetrazine used has limited stability to aqueousmedia. (Blackman et al., 2008, J Am Chem Soc, 130, 13518-9; Devaraj etal., 2009, Angew Chem Int Ed Engl, 48, 7013-6; Devaraj et al., 2008,Bioconjug Chem, 19, 2297-9; Pipkorn et al., 2009, J Pept Sci, 15,235-41).

In some embodiments, the detectable agent carries a functional groupsuch as an amine, alcohol, carboxylic acid or ester, or other group ofatoms that can undergo a chemical reaction allowing attachment to thediene or dienophile. Alternatively or in addition, the dienophile orheterodienophile (which can be, e.g., an alkene, alkyne, nitroso,carbonyl or imine) possesses a reactive functional group for attachmentto the detectable agent. Thus, the reactive functional group on thedetectable agent and/or diene/dienophile undergoes a chemical reactionto form a link between the detectable agent and the diene or dienophile.

In some embodiments, the diene or dienophile can be incorporated into aPARP1 inhibitor and/or a detectable agent. In some embodiments, thediene or dienophile is incorporated through a linker. One of skill inthe art could readily synthesize such compounds using known syntheticmethods or modifying the examples provided herein.

In some embodiments, the diene can be a substituted tetrazine or otherheteroaromatic ring system with at least two nitrogens adjacent to eachother, and which is a highly reactive participant in the inverseelectron demand Diels-Alder reaction. The diene can be linked to theamino acid or unnatural amino acid through the use of a linker. In thisembodiment, the diene possesses a reactive group such as an amine,alcohol, carboxylic acid, ester, or activated ester, or other group thatcan undergo a chemical reaction with the reactive moiety on the PARP1inhibitor or the detectable agent to form a link between the two. Dienesuseful in the present disclosure include but are not limited to aromaticring systems that contain two adjacent nitrogen atoms, for example,tetrazines, pyridazines, substituted or unsubstituted 1,2-diazines.Other 1,2-diazines can include 1,2-diazines annelated to a secondπ-electron-deficient aromatic ring such as pyrido[3,4-d]pyridazines,pyridazino[4,5-d]pyridazines, and 1,2,4-triazines. Pyridazines can alsobe fused with a five-membered heterocycle such asimidazo[4,5-d]pyridazines and 1,2,3-triazolo[4,5-d]pyridazines. In someembodiments, the diene is an asymmetrical tetrazine as described herein,e.g., 3-(p-Benzylamino)-1,2,4,5-tetrazine (Structure I).

Dienophiles useful in the present methods and compositions include butare not limited to carbon containing dienophiles such as alkenes oralkynes, or compounds containing nitroso, carbonyl or imine groups. Insome embodiments, the dienophile is a strained dienophile. As usedherein, a “strained” dienophile has a dihedral angle that deviates fromthe idealized 180 degree dihedral angle. Alternatively, non-straineddienophiles (e.g., sytrenes) and/or electron rich electrophiles (e.g.,eneamines or vinyl ethers), can also be used with nitroso compounds.Alkenes as used herein refer to an alkyl group having one or more doublecarbon-carbon bonds such as an ethylene, propylene, and the like.Alkenes can also include cyclic, ring-strained alkenes such astrans-cyclooctene or norbornene carrying a double bond which inducessignificant ring strain and is thus highly reactive. Alkenes can alsoinclude more complex structures such as indoles and azaindoles, electronrich enamines. Heterodienophiles containing carbonyl, nitroso or iminegroups can also be used. In some embodiments, the dienophile is atrans-cyclooctene or a trans-cyclooctenol, e.g., (E)-cyclooct-4-enol.

In some embodiments, the detectable agent is chemically attached to thedienophile. For example, the strained alkene is chemically coupled to adetectable agent, e.g., a trans-cyclooctene modified with a radioactiveisotope, e.g., ¹⁸F (see Structure II below).

Applicable to all other NHS esters, peptide bond formation is alsopossible using other standard coupling techniques (e.g. EDCI, DCI,HOBT/TBTU, and others).

In some embodiments, AZD2281-BODIPY FL is reacted with trialkylsilyltriflates (e.g. trimethylsilyl triflate) and in some cases followed bydialkylaminopyridines (e.g. dimethylaminopyridine, DMAP). ¹⁸F-Fluorideis added and the ¹⁸F-AZD2281-BODIPY FL is being produced through theexchange of triflate or DMAP at the BODIPY boron core with ¹⁸F-fluoride.In some embodiments, trialkylsilyls triflates are immobilized on a solidsupport (e.g. magnetic materials, dextrans, polystyrenes, latex,biological macromolecules). In some embodiments, 4-amino pyridines areimmobilized on a solid support (e.g. magnetic materials, dextrans,polystyrenes, latex, biological macromolecules). The reactive solidsupport is then treated with AZD2281-BODIPY FL. Incubation with¹⁸-fluoride is followed by filtration of the solid support, resulting inhigh specific activity labeled material (e.g. high specific activityPARP1 imaging agents). See, for example, FIG. 15.

One problem during the synthesis of ¹⁸F-radiolabeled compounds is thatin most cases large amounts of precursors are used to efficiently reactwith small quantities of ¹⁸F. The resulting mixtures can be purified byHPLC to remove the excess starting material, which in most cases willcompete with the radiolabeled probe for the targeted binding sites. Insome embodiments, however, to avoid lengthy HPLC purifications, theresulting materials can be purified using a scavenger resin as providedherein. The resins can include those of Formula (3):

R—NH-L-X

wherein:R is a resin bead;L is a linker; andX is a moiety that reacts with the unreacted starting material.

The resin bead can be made of many types of materials known to those ofskill in the art as long as the bead is large enough to be filtered. Insome embodiments, the resin bead can be magnetic, a modified filter, amodified column material, etc. For example, the resin can be made ofdextran, silica, glass, or any other non-reactive solid support. In someembodiments, the resin bead is a magnetic resin bead.

In some embodiments, X is a trans-cyclooctene. For example, anon-limiting example of a resin includes:

Accordingly, provided herein is a method of purifying a compositioncomprising an ¹⁸F-labeled compound (e.g., a compound of Formula (1). Themethod can include contacting a composition comprising a ¹⁸F-labeledcompound and excess of non-labeled starting material with an excess of aresin of Formula (3); and separating out the product of the startingmaterial and the resin of Formula (3). In some embodiments, the methodcan result in a composition comprising at least about 75% of an¹⁸F-labeled compound (e.g., at least about 80%, at least about 85%, atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, and at least about 99%). Aresin of Formula (3) can be prepared, for example, by reaction ofcommercially available magnetic amine-decorated beads in a solution ofDMF/PBS with a 75 mm solution of NHS-activated trans-cyclooctene (seeFIG. 5).

The reactions for preparing the compounds provided herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds provided herein can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in Protecting GroupChemistry, 1^(st) Ed., Oxford University Press, 2000; March's AdvancedOrganic chemistry: Reactions, Mechanisms, and Structure, 5^(th) Ed.,Wiley-Interscience Publication, 2001; and Peturssion, S. etal.,“Protecting Groups in Carbohydrate Chemistry,” J. Chem. Educ.,74(11), 1297 (1997) (each of which is incorporated herein by referencein their entirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible,fluorescence), mass spectrometry, or by chromatographic methods such ashigh performance liquid chromatography (HPLC), liquidchromatography-mass spectroscopy (LCMS), thin layer chromatography(TLC), or radio-thin layer chromatograph (rTLC). Compounds can bepurified by those skilled in the art by a variety of methods, includinghigh performance liquid chromatography (HPLC) (“Preparative LC-MSPurification: Improved Compound Specific Method Optimization” K. F.Blom, et al., J. Combi. Chem. 6(6) (2004), which is incorporated hereinby reference in its entirety) and normal phase silica chromatography.

Pharmaceutical Compositions

The methods provided herein include the manufacture and use ofpharmaceutical compositions, which include compounds identified by amethod provided herein as active ingredients. Also included are thepharmaceutical compositions themselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration.

A pharmaceutical composition is typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection can include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The composition should be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration ofadministration may be determined empirically using known testingprotocols or by extrapolation from in vivo or in vitro test data. It isto be noted that concentrations and dosage values may also vary with thesize of the area to be imaged. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

Dosage forms or compositions containing a compound as described hereinin the range of 0.005% to 100% with the balance made up from non-toxiccarrier may be prepared. Methods for preparation of these compositionsare known to those skilled in the art. The contemplated compositions maycontain 0.001%-100% active ingredient, in one embodiment 0.1-95%, inanother embodiment 75-85%.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Use

The compounds described herein can be imaged using methods known in theart. For example, imaging can be achieved in living animals, organs, ortissues, using e.g. near infrared (NIR), MR imaging (MRI), positronemission tomography (PET), single photon computerized tomography(SPECT), or other whole body imaging modalities. The detectable agent ofthe compound can be imaged by these whole body imaging modalities todetect cancer cells (e.g., cancer cells overexpressing PARP1). Forexample, a compound having a fluorescent detectable agent can bedetected by traditional fluorescence imaging techniques allowing for thefacile tracking of the compounds by fluorescence microscopy or flowcytometry using methods known in the art, e.g., as described in US2005/0249668, the content of which is incorporated by reference in itsentirety. In some embodiments, a compound having a radioactive agent canbe imaged using positron emission tomography (PET).

The compositions and methods described herein can be imaged using avariety of modalities that are known to one of skill in the art.Detection methods can include both imaging ex vivo and in vivo imagingmethods, e.g., immunohistochemistry, bioluminescence imaging (BLI),Magnetic Resonance Imaging (MM), positron emission tomography (PET),Single-photon emission computed tomography (SPECT), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required. In some embodiments, one or moreimaging techniques can be used in the methods provided herein. Forexample, fluorescence, PET and/or SPECT imaging can be used.

The compounds and compositions described herein can be used in in vivoimaging methods to detect, quantify and evaluate PARP1 (e.g., PARP1 in acancer). In general, such methods include administering to a subject oneor more compounds of Formula (1) or Formula (2) described herein;optionally allowing the compound to distribute within the subject; andimaging the subject, e.g., by fluoroscopy, radiography, computedtomography (CT), MM, PET, SPECT, laparoscopy, endomicroscopy, or otherwhole body imaging modality to detect the presence of PARP1.Furthermore, it is understood that the methods (or portions thereof) canbe repeated at intervals to evaluate the subject and detect any changesin PARP1 concentration over time. Information provided by such in vivoimaging, for example, the presence, absence, or level of emitted signal,can be used to detect and/or monitor the loss of PARP1 or increase ofPARP1, e.g., after medical treatment.

A number of preclinical and clinical applications for a compoundprovided herein can be envisioned. For example, a compound describedhere can be used: 1) for the early detection cancers (e.g., cancers thatoverexpress PARP1 such as pancreatic cancer); 2) as an aid to surgeonsduring surgery (e.g., by allowing for real-time detection of cancercells); and 3) as a method for monitoring the progress of a cancertreatment (e.g., by quantifying the amount of PARP1 present before,during, and after treatment).

In some embodiments, the methods provided herein include methods fordetecting a cancer, i.e., a cancer that overexpresses PARP1. Generally,the methods include administering an effective amount of a compound(i.e., active ingredient) as provided herein (i.e., a compound ofFormula (1)) to a subject who is in need of, or who has been determinedto be in need of, such detection and detecting the compound. Inaddition, provided herein are methods for imaging a cancer cell.Generally, the methods include contacting a cell with an effectiveamount of a compound as provided herein and imaging the cell. Suchmethods may be conducted in vitro or in vivo.

The compounds according to Formula (1) can be used to detect any cancerthat overexpresses PARP1, are believed effective to detect and image abroad range of cancers types, including but not limited to breastcancer, ovarian cancer, and prostate cancer. In some embodiments, thecancer is an epithelial cancer. The methods and compositions describedherein can be used to help a physician or surgeon to identify andcharacterize cancers. The methods and compositions described herein canalso be used in the detection, characterization, and/or determination ofthe localization of the cancer, especially early in the disease, theseverity of a cancer, the staging of a cancer, and/or monitoring acancer. The presence, absence, or level of an emitted signal can beindicative of the state of the cancer.

Cancers that may be detected and/or imaged by the compounds,compositions and methods described herein include, but are not limitedto, the following:

-   -   cardiac cancers, including, for example sarcoma, e.g.,        angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma;        myxoma; rhabdomyoma; fibroma; lipoma and teratoma;    -   lung cancers, including, for example, bronchogenic carcinoma,        e.g., squamous cell, undifferentiated small cell,        undifferentiated large cell, and adenocarcinoma; alveolar and        bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma;        chondromatous hamartoma; and mesothelioma;    -   gastrointestinal cancer, including, for example, cancers of the        esophagus, e.g., squamous cell carcinoma, adenocarcinoma,        leiomyosarcoma, and lymphoma; cancers of the stomach, e.g.,        carcinoma, lymphoma, and leiomyosarcoma; cancers of the        pancreas, e.g., ductal adenocarcinoma, insulinoma, glucagonoma,        gastrinoma, carcinoid tumors, and vipoma; cancers of the small        bowel, e.g., adenocarcinoma, lymphoma, carcinoid tumors,        Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,        and fibroma; cancers of the large bowel, e.g., adenocarcinoma,        tubular adenoma, villous adenoma, hamartoma, and leiomyoma;    -   genitourinary tract cancers, including, for example, cancers of        the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma),        lymphoma, and leukemia; cancers of the bladder and urethra,        e.g., squamous cell carcinoma, transitional cell carcinoma, and        adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma,        and sarcoma; cancer of the testis, e.g., seminoma, teratoma,        embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,        interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid        tumors, and lipoma;    -   liver cancers, including, for example, hepatoma, e.g.,        hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma;        angiosarcoma; hepatocellular adenoma; and hemangioma;    -   bone cancers, including, for example, osteogenic sarcoma        (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,        chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum        cell sarcoma), multiple myeloma, malignant giant cell tumor        chordoma, osteochrondroma (osteocartilaginous exostoses), benign        chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma        and giant cell tumors;    -   nervous system cancers, including, for example, cancers of the        skull, e.g., osteoma, hemangioma, granuloma, xanthoma, and        osteitis deformans; cancers of the meninges, e.g., meningioma,        meningiosarcoma, and gliomatosis; cancers of the brain, e.g.,        astrocytoma, medulloblastoma, glioma, ependymoma, germinoma        (pinealoma), glioblastoma multiform, oligodendroglioma,        schwannoma, retinoblastoma, and congenital tumors; and cancers        of the spinal cord, e.g., neurofibroma, meningioma, glioma, and        sarcoma;    -   gynecological cancers, including, for example, cancers of the        uterus, e.g., endometrial carcinoma; cancers of the cervix,        e.g., cervical carcinoma, and pre tumor cervical dysplasia;        cancers of the ovaries, e.g., ovarian carcinoma, including        serous cystadenocarcinoma, epithelial cancer, mucinous        cystadenocarcinoma, unclassified carcinoma, granulosa thecal        cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and        malignant teratoma; cancers of the vulva, e.g., squamous cell        carcinoma, intraepithelial carcinoma, adenocarcinoma,        fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear        cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and        embryonal rhabdomyosarcoma; and cancers of the fallopian tubes,        e.g., carcinoma;    -   hematologic cancers, including, for example, cancers of the        blood, e.g., acute myeloid leukemia, chronic myeloid leukemia,        acute lymphoblastic leukemia, chronic lymphocytic leukemia,        myeloproliferative diseases, multiple myeloma, and        myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's        lymphoma (malignant lymphoma) and Waldenström's        macroglobulinemia;    -   skin cancers, including, for example, malignant melanoma, basal        cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles        dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,        psoriasis; and    -   adrenal gland cancers, including, for example, neuroblastoma.

Cancers may be solid tumors that may or may not be metastatic. Cancersmay also occur, as in leukemia, as a diffuse tissue. Thus, the term“cancer cell”, as provided herein, includes a cell afflicted by any oneof the above identified disorders.

The compounds according to Formula (1) can also be administered to asubject in combination with surgical methods to treat cancers, e.g.,resection of tumors. The compounds can be administered to the individualprior to, during, or after the surgery. The compounds can beadministered parenterally, intravenous or injected into the tumor orsurrounding area after tumor removal, e.g., to image or detect residualcancer cells. For example, the compound may be used to detect thepresence of a tumor and to guide surgical resection. In someembodiments, the compound can be used to detect the presence of residualcancer cells and to guide continued surgical treatment until at least aportion (e.g., all) such cells are removed from the subject.Accordingly, there is provided a method of guided surgery to remove atleast a portion of a tumor from a subject comprising providing acompound of Formula (1); causing the compound to be present in at leastsome cancer cells in an effective amount to inhibit PARP1 and fordetection of the compound to be observable; observing the image (e.g.,fluorescence, PET or SPECT scan); and performing surgery on the subjectto remove at least a portion of the tumor that comprises detected cancercells.

In some embodiments, a compound of Formula (1) or Formula (2) is imagedin vivo using PET or SPECT imaging. For example, the use of such methodspermits the facile, real-time imaging and localization of cancerslabeled with a compound having a radioactive detectable agent. In someembodiments, PET or SPECT can be used to diagnose cancer by imaging theaccumulation of a compound provided herein in cancer cells expressingPARP1. In some embodiments, the use of PET and/or SPECT as an imagingmodality can be useful during surgery to locate cancer cells (e.g.,residual cancer cells following excision of a tumor mass).

In some embodiments, a compound of Formula (1) or Formula (2) is imagedin vivo using laparoscopy and endomiscroscopy. For example, the use oflaparoscopy permits the facile, real-time imaging and localization ofcancers labeled with a compound having a fluorescent detectable agent.In some embodiments, laparoscopy can be used to diagnose cancer byimaging the accumulation of a compound provided herein in cancer cellsexpressing PARP1. In some embodiments, the use of laparoscopy as animaging modality can be useful during surgery to locate cancer cells(e.g., residual cancer cells following excision of a tumor mass). Insome embodiments, a compound can be imaged using fiber opticendomicroscopy.

In addition, in vivo imaging can be used to assess the effect of ananti-cancer therapy on cells expressing PARP1, by using the compoundsdescribed herein, wherein the subject is imaged prior to, during, and/orafter treatment with the therapy, and the corresponding signal/imagesare compared. For example, a subject with a cancer can be imaged priorto and after treatment with chemotherapy or radiation therapy todetermine the response of the PARP1-expressing cancer cells totreatment.

As described herein, a compound of Formula (1) accumulates in PARP1expressing cells (e.g., PARP1 overexpressing cancer cells). Thisaccumulation, however, can be quantifiably inhibited by administrationof an unlabeled PARP1 inhibitor, e.g., the parent PARP1 inhibitorpresent in the compound of Formula (1) or any other PARP inhibitor.Accordingly, further provided herein is a method for measuring PARP1inhibition by any PARP1 inhibitor or compound that has an effect on PARPexpression or activity level. Generally, the method includes contactinga cell (e.g., administering to a subject) an effective amount of acompound of Formula (1) and a therapeutic PARP1 inhibitor (i.e.,non-labeled), and imaging the cell (e.g., the subject) to determine theamount of the compound of Formula (1) present.

In addition to promising approaches of PARP1 inhibitors as anti-cancerdrugs, it has been shown that DNA-damage leads to rapid activation (10-to 500-fold) and recruitment of PARP1 (see, e.g., Hassa, 2008, Front.Biosci., 13, 3046-3082; Jean-François Haince and Michael J. Hendzel,2008, J. Biol. Chem., 283, 1197-1208). Although not much is known aboutPARP1 protein levels and activities in tumor cells versus healthy cells(see Michele Rouleau and Guy G. Poirier, 2010, Nature Rev Cancer, 10,293-301), different reports show increased activity and/or expressionlevels of PARP1 in cancer cell lines and tumors versus healthy tissue(Zaremba T, 2009, Br J Cancer., 101, 256-262; Katsuhiko Nosho, 2006,Eur. J. Cancer, 42, 2374-2381).

With respect to in vitro imaging methods, the compounds and compositionsdescribed herein can be used in a variety of in vitro assays. Anexemplary in vitro imaging method comprises: contacting a sample, forexample, a biological sample (e.g., a cell such as a cancer cell), withone or more compounds of Formula (1) or Formula (2); allowing theconjugate(s) to interact with a biological target in the sample;optionally, removing unbound agents; illuminating the sample with lightof a wavelength absorbable by a fluorophore of the agents; and detectinga signal emitted from fluorophore thereby to determine whether the agenthas been activated by or bound to the biological target.

After a compound has been designed, synthesized, and optionallyformulated, it can be tested in vitro by one skilled in the art toassess its biological and performance characteristics. For instance,different types of cells grown in culture can be used to assess thebiological and performance characteristics of the compound. Cellularuptake, binding or cellular localization of the agent can be assessedusing techniques known in the art, including, for example, fluorescentmicroscopy, fluorescence-activated cell sorting (FACS) analysis,immunohistochemistry, immunoprecipitation, in situ hybridization andForster resonance energy transfer (FRET) or fluorescence resonanceenergy transfer.

By way of example, the compound can be contacted with a sample for aperiod of time and then washed to remove any free compound. The samplecan then be viewed using an appropriate detection device such as afluorescent microscope equipped with appropriate filters matched to theoptical properties of a fluorescent agent. Fluorescence microscopy ofcells in culture or scintillation counting is also a convenient meansfor determining whether uptake and binding has occurred. Tissues, tissuesections and other types of samples such as cytospin samples can also beused in a similar manner to assess the biological and performancecharacteristics of the compounds. Other detection methods including, butnot limited to flow cytometry, immunoassays, hybridization assays, andmicroarray analysis can also be used.

In some embodiments, the compounds can be used in an in vitro assay fordetecting agents that inhibit PARP1.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims. Compoundnumbers are independent in each of the following Examples.

Example 1 A. Preparation of AZD2281-TCO (7) Materials and Methods

Until otherwise noted, all reagents were purchased from Sigma-Aldrich(St. Louis, Mo.) and used without further purification. Texas Red-X,succinimidyl ester was purchased from Invitrogen (Carlsbad, Calif.).Cyclohexylcarbodiimide polystyrene resin was purchased from EMDbiosciences (Gibbstown, N.J.). LC-ESI-MS analysis and HPLC purificationswere performed on a Waters (Milford, Mass.) LC-MS system. For LC-ESI-MSanalyses, a Waters XTerra® C18 5 μm column was used. For preparativeruns, an Atlantis® Prep T3 OBD™ 5 μM column was used. High-resolutionelectrospray ionization (ESI) mass spectra were obtained on a BrukerDaltonics APEXIV 4.7 Tesla Fourier Transform mass spectrometer(FT-ICR-MS) in the Department of Chemistry Instrumentation Facility atthe Massachusetts Institute of Technology. Cellular images were taken ona Nikon (Tokyo, Japan) Eclipse 80i microscope with either a Nikon PlanApo 40×/0.95 air or a Nikon Plan Apo 60×/1.45 oil immersion objective.IC₅₀ assays were analyzed using a Tecan (Männedorf, Switzerland) Safiremicroplate system. All kinetic data were analyzed using Prism 4(GraphPad, La Jolla, Calif.) for Mac.

General Methods

Texas Red-Tz (8) was synthesized similar to methods described earlier.(See N. K. Devaraj et al., Angew. Chem., Int. Ed. 2010, 49, 2869-2872;N. K. Devaraj et al., Angew. Chem., Int. Ed. 2009, 48, 7013-7016,S7013/1-S7013/6; and N. K. Devaraj et al., Bioconj. Chem. 2008, 19,2297-2299.(Cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one(1),4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(2) and (E)-cyclooct-4-enyl 2,5-dioxopyrrolidin-1-yl carbonate (5) weresynthesized as described earlier. (See K. A. Menear et al., J. Med.Chem. 2008, 51, 6581-6591; and N. K. Devaraj et al., Angew. Chem., Int.Ed. 2009, 48, 7013-7016, S7013/1-S7013/6).

Preparation of4-[[4-Fluoro-3-(4-(5-oxopentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(3)

Glutaric anhydride (0.50 g, 4.37 mmol) and N,Ndiisopropylethylamine(2.28 mL, 13.11 mmol) were added to a solution of4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (2) (1.60 g, 4.37 mmol) in dichloromethane(50 mL) and the reaction mixture and stirred for 30 minutes. Water (50mL) was then added and the reaction mixture stirred for another 30minutes. The reaction mixture was acidified with HCl to pH 2, theorganic phase separated and the aqueous phase extracted withdichloromethane (3×30 mL). The combined organic phases were dried overMgSO₄ and volatiles removed in vacuo. The resulting crude material waspurified using silica chromatography (0%-30% MeOH/DCM), yielding thepure product as an off-white solid (1.52 g, 3.16 mmol, 72%).

Preparation of4-[[4-Fluoro-3-(4-(N-(2-aminoethyl)-5-oxo-pentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(4)

Cyclohexylcarbodiimide polystyrene resin (634 mg, 2.3 mmol/g) was addedto a solution of4-[[4-Fluoro-3-(4-(5-oxopentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(3) (350 mg, 0.73 mmol) in dichloromethane (20 mL) and the resultingmixture stirred gently for 7 hours at room temperature. Subsequently,ethylenediamine (976 μL, 14.6 mmol) was added and the reaction mixturestirred for another 60 minutes, before the reaction mixture was filteredand volatiles removed in vacuo. The crude material was purified viaHPLC, yielding the title compound as a clear solid (62 mg, 0.12 mmol,16%).

Preparation of AZD2281-NOB (6)

Cyclohexylcarbodiimide polystyrene resin (33 mg, 2.3 mmol/g) andtriethylamine (16 μL, 0.11 mmol) were added to a solution of4-[[4-Fluoro-3-(4-(N-(2-aminoethyl)-5-oxo-pentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (4) (20 mg, 38 μmol) and5-norbornene-2-carboxylic acid (11 mg, 77 μmol) in dichloromethane (1mL) and the resulting mixture stirred gently over night at roomtemperature. Subsequently, the reaction mixture was filtered andvolatiles removed in vacuo. The crude material was purified via HPLC,yielding the title compound as a clear solid (5.9 mg, 9 24%).

Preparation of AZD2281-TCO (7)

Triethylamine (16 μL, 0.11 mmol) was added to a solution of4-[[4-Fluoro-3-(4-(N-(2-aminoethyl)-5-oxo-pentanamide)piperazine-1-carbonyl)phenyl]methyl]-2Hphthalazin-1-one (4) (20 mg, 38 μmol) and(E)-cyclooct-4-enyl-2,5-dioxopyrrolidin-1-yl carbonate (5) (12 mg, 46μmol) in dichloromethane (1 mL) and the resulting mixture stirred gentlyover night at room temperature. Subsequently, the reaction mixture wasfiltered and volatiles removed in vacuo. The crude material was purifiedvia HPLC, yielding the title compound as a clear solid (11.3 mg, 1744%).

B. HPLC Characterization of Reaction Between TexasRed-Tetrazine (8) andAZD2281-TCO (7)

AZD2281-TCO (7) and Texas Red-tetrazine (8) were combined in 40 μL ofDMSO/PBS at a final concentration of 0.3 mM for each reagent. Thesolution was stirred for several minutes at room temperature, yieldingAZD2281-Texas Red (9). See FIG. 1. FIG. 1A shows the HPLC trace for theAZD2281-Texas Red (9) crude reaction mixture. LC-MS spectra confirmedthe quantitative conversion of Texas Red-Tz (8). Multiple peaks wereidentified with a molecular mass corresponding to AZD2281-Texas Red (9;FIG. 1 B, m/z 1536.0 [M+H]+). These were the result of different isomersformed in the tetrazine trans-cyclooctene cycloaddition. The fast andselective conversion of AZD2281-TCO (7) to AZD2281-Texas Red (9) in thepresence of Texas Red-Tz (8) indicates that these small molecules alsohave potential applicability to in vivo experiments.

C. PARP-1 IC₅₀ Determination

The inhibitory potentials of AZD2281 derivatives 6 and 7, and ofpre-reacted AZD2281-Texas Red (9), were tested using a PARP1 activityassay. A commercially available colorimetric assay (Trevigen,Gaithersburg, Md.) was used to measure PARP activity in vitro in thepresence of inhibitors. Ten-fold dilutions of AZD2281-derivatives (6)(final concentration 400 nM to 0.04 nM); and (7), (9) (4 μM to 0.04 nM)were incubated with 0.5 units PARP HSA for 10 minutes in histone-coated96-well plates. All experiments were carried out in triplicate. Controlsamples did not contain inhibitor and background measurement samples didnot contain PARP-1. All reaction mixtures were adjusted to a finalvolume of 50 μL and a maximum final concentration 0.4% DMSO in assaybuffer. The remainder of the assay was performed according to themanufacturer's instructions. PARP activity was measured by absorbance at450 nm in each well using a Safire 2 microplate reader (Tecan Group,Mannedorf, Switzerland). IC₅₀ values were calculated using Prismsoftware (GraphPad, La Jolla, Calif.).

Analysis of the AZD2281 derivatives 6 and 7 resulted in IC₅₀ values of10.1±1.3 nM and 11.8±1.4 nM, respectively. Thus, modification andconjugation of linkers and fluorophores to the 4-NH-piperazine group ofAZD2281 precursor 2 appear to be tolerated by the enzyme, and allow thedesign of bifunctional derivatives. The IC₅₀ value of pre-reactedAZD2281-Texas Red (9; 15.4±1.2 nM) demonstrates that thetrans-cyclooctene/tetrazine cycloaddition only minimally reduces bindingof the 1(2H)-phthalazinone to PARP1, which confirms its possibleapplication as an imaging probe.

D. In Vitro Cell Assays

Due to their fast reaction kinetics compared to AZD2281-NOB (6), thetrans-cyclooctene conjugated AZD2281 (AZD2281-TCO; 7) and Texas Red-Tz(8) were tested under in vivo conditions in live cells.

Cell Culture

MDA-MB-231 and MDA-MB-436 cells were obtained from the ATCC and culturedin RPMI 1640 supplemented with 10% fetal bovine serum, L-glutamine, andpenicillin/streptomycin. MDA-MB-231 and MDA-MD-436 cells stablyexpressing PARP-GFP were derived by transfection with Lipofectamine 2000(Invitrogen) and isolation of individual G418-resistant clones. Cellswere maintained in growth medium containing 1 mg/mL G418. Expression ofPARP-GFP in MDA-MB-436 cells was verified using immunofluorescencemicroscopy to show nuclear localization of PARP-GFP, and westernblotting for PARP-GFP.

For AZD2281-TCO IC₅₀ assays, MDA-MB-436 cells (500 μL, 80.000 cells/mL)were seeded into glycerine-treated 8-well chamber slides (Lab Tek™,Thermo Scientific, Rochester, N.Y.), and allowed to attach overnight.Cells were then incubated with Texas Red-Tz (8) (25 μL, 20 μM) for 20minutes (37° C.) before the medium was removed and cells were washed(1×, medium, 500 μL). Subsequently, 500 μL medium, Hoechst 33258 (10 μL,100× in PBS) and AZD2281-TCO (7) (25 μL, in PBS, 3% DMSO, 30 μM-100 nM)were added and incubated at 37° C. for 20 minutes. Cells were washedwith PBS (3×500 μL), fixed with paraformaldehyde (4% in PBS) and washedwith PBS (3×500 μL, time between each wash=5 minutes). PBS was removedand cells mounted using Prolong Gold (Invitrogen, Carlsbad, Calif.)before imaging.

For AZD2281-TCO blocking experiments, MDA-MB-436 cells (500 μL, 80.000cells/mL) were seeded into glycerine-treated 8-well chamber slides (LabTek™, Thermo Scientific, Rochester, N.Y.), and allowed to attachovernight. Cells were then incubated with Texas Red-Tz (8) (25 μL, 20μM) for 20 minutes (37° C.) before the medium was removed and cells werewashed (1×, medium, 500 μL). Subsequently, 500 μL medium, Hoechst 33258(10 μL, 100× in PBS), AZD2281 (1) (5 μL in DMSO, 10 mM) and AZD2281-TCO(7) (25 μL, in PBS, 3% DMSO, 30 μM-100 nM) were added and incubated at37° C. for 20 min. Cells were washed with PBS (3×500 μL), fixed withparaformaldehyde (4% in PBS) and washed with PBS (3×500 μL, time betweeneach wash=5 min). PBS was removed and cells mounted using Prolong Gold(Invitrogen, Carlsbad, Calif.) before imaging.

Image Analysis

Cells were observed on a Nikon 80i (Nikon, Tokyo JP) microscope equippedwith an ImagEM camera (Hammamatsu Photonics, Tokyo JP). Images detailingdistinct and separate cellular regions were obtained with the followingfilters: (Dapi, excitation 350±50 nm, emission 460±50 nm, dichroic400LP; IgG Pab, excitation 480±20 nm, emission 535±25 nm, dichroic505LP; Texas Red-Tz (8), excitation 560±20 nm, emission 630±30 nm,dichroic 595DCLP; Chroma Technology, Bellows Falls, Vt.). Images in eachchannel were captured using identical acquisition parameters. For eachimage, both cell structures and nuclei structures have been obtainedusing the appropriate fluorescence filters and appropriate excitationsignal levels to avoid collecting auto-fluorescence. The collected datawas then pre-processed with Cellprofiler (A. E. Carpenter, et al.,GenomeBiology 2006, 7, R100) and statistics collected using Matlab (TheMathworks, Waltham, Mass.). Cell structures were then identified usingOtsu's method (N. Malpica, et al., Cytometry 1997, 28, 289-297; and N.Otsu, IEEE Trans. Sys. Man. Cyber. 1979, 9, 62-66) and binary masks weregenerated matching the boundary of each cell structure. Within eachimage and for each cell, the fluorescence signal for the total cell andthe signal in the nuclear area was calculated using the correspondingmask as a spatial filter. The two signals were then normalized for theirtotal areas. The ratio of the fluorescent signal in the cytosol regionover the signal in the nuclear region was then calculated. Backgroundsubtraction was performed on the normalized signal using the cell'snegative masks. For all cells we chose an optimal ratio between 0.35 and0.48 for the cytosol area over the nucleus area.

MDA-MB436 Cellular Imaging

MDA-MB-436 cells (500 μL, 80.000 cells/mL) were seeded intoglycerine-treated 8-well chamber slides (Lab Tek™, Thermo Scientific,Rochester, N.Y.), and allowed to attach overnight. They were incubatedwith Texas Red-Tz (8) (25 μL, 20 μM) for 20 minutes (37° C.) before themedium was removed and cells were washed (1×, medium, 500 μL).Subsequently, 500 μL medium and AZD2281-TCO (7) (25 μL, in PBS, 3% DMSO,30 μM) were added and incubated at 37° C. for 20 min. Cells were washedwith PBS (3×500 μL), fixed with paraformaldehyde (4% in PBS) and washedwith PBS (3×500 μL, time between each wash=5 min). Cells werepermeabilized using Triton-X-100 (2% in PBS, 10 min) and incubated withanti-PARP-1 Mab (EMD biosciences, Gibbstown, N.J.) for 3 hours, beforestained with secondary IgG-GFP Pab (EMD biosciences, Gibbstown, N.J.).PBS was removed and cells mounted using Prolong Gold (Invitrogen,Carlsbad, Calif.) before imaging.

Results

Staining patterns for the Texas Red dye (FIG. 2A) showed that it notonly localized in the nucleus, but also accumulated in the nucleolus.Whilst anti-PARP1 Mabs showed similar nuclear localization (FIG. 2B),there was an absence of Mab signal in the nucleoli. This was presumablydue to steric hindrance and not to the absence of PARP in the nucleolus,as earlier reports have indicated (V. S. Meder et al., J. Cell Sci.2005, 118, 211-222; and J.-F. Haince et al., J. Biol. Chem. 2008, 283,1197-1208). The results in FIG. 2C confirm that there was excellentspatial correlation between the small molecules (AZD2281-TCO (7)/TexasRed-Tz (8)).

E. PARP1-GFP Reporter Construct

A PARP1-GFP fusion protein was also constructed and expressed inMDA-MB436 cells as an independent confirmation of probe localization.PARP-GFP was constructed by PCR of human PARP-1 from Open Biosystemsclone 5193735 from the NIH_MGC_114 cDNA library, and cloning intopAcGFP-N1 between XhoI and XmaI restriction sites. The PARP-GFPconstruct was verified by DNA sequencing.

PARP-1-GFP MDA-MB436 Cellular Imaging

PARP-1-GFP expressing MDA-MB-436 cells (500 μL, 80.000 cells/mL) wereseeded into glycerine-treated 8-well chamber slides (Lab Tek™, ThermoScientific, Rochester, N.Y.), and allowed to attach overnight. They werewashed with PBS (3×500 μL), fixed with paraformaldehyde (4% in PBS) andwashed with PBS (3×500 μL, time between each wash=5 min). Cells werepermeabilized using Triton-X-100 (0.5% in PBS, 10 min) and incubatedwith anti-PARP-1 Mab (EMD biosciences, Gibbstown, N.J.) for 3 h, beforestained with secondary IgG-GFP Pab (EMD biosciences, Gibbstown, N.J.).PBS was removed and cells mounted using Prolong Gold (Invitrogen,Carlsbad, Calif.) before imaging.

In the PARP1-GFP expressing cells, GFP expression was clearly observed,primarily in the nucleoli but also in the nucleus. This pattern wasidentical to that seen with the AZD2281-TCO/Texas Red-Tz pair. (Table 1,FIG. 3). Interestingly, incubation of live cells with Texas Red-Tz (8)without AZD2281-TCO (7) did not lead to nuclear localization of the dye.Instead, there was non-lower nuclear/cytoplasmic localization ratios(modest increase of 38±2%). The superior results of sequential treatmentof MDAMB436 cells with AZD2281-TCO (7) and Texas Red-Tz (8) demonstratesthe advantages of a bioorthogonal in vivo reaction, since both partners(the targeting molecule 7 as well as the fluorophore 8) are small insize (7: 674.76 gmol⁻¹; 8: 889.05 gmol⁻¹), and this contributes toeasier permeation. Once assembled, however, penetration is lessefficient leading to trapping of the conjugate and hightarget-to-background ratios.

TABLE 1 IC₅₀ values for the bifunctional 1(2H)-phthalazinone- basedtargeted probes and for AZD2281-Texas Red. MW IC₅₀ Reactive CompoundName # [gmol⁻¹] [nm] with Reactive AZD2281-NOB 6 642.7 10.1 + 1.3Tz-fluorochromes InhibitorAZD2281-TCO 7 674.8 11.8 + 1.4Tz-fluorochromes Fluorescent AZD2281-Texas Red 9 1535.6 15.4 + 1.2 NAInhibitor

Example 2 A. Synthesis Materials and Methods.

Unless otherwise noted, all reagents were purchased from Sigma-Aldrich(St. Louis, Mo.) and used without further purification.Cyclohexylcarbodiimide polystyrene resin was purchased from EMDbiosciences (Gibbstown, N.J.).4-[[4-Fluoro-3-(4-(5-oxopentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (7), tetrazine amine (8), and4-[[4-fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(11) were synthesized as described earlier (T. Reiner et al.,ChemBioChem 2010; N. K. Devaraj et al., Bioconjug. Chem. 2008, 19,2297-2299; and K. A. Menear et al., J. Med. Chem. 2008, 51, 6581-6591.¹⁸F-Fluoride (n.c.a.) in ¹⁸O-enriched water was purchased from PETNET(Woburn, Mass.). Automated synthesis of ¹⁸F-labeled trans-cyclooctenewas carried out using a Synthra RN Plus automated synthesizer (SynthraGmbH, Hamburg, Germany) operated by SynthraView software. Fornonradioactive compounds, LC-ESI-MS analysis and HPLC-purifications wereperformed on a Waters (Milford, Mass.) LC-MS system. For LC-ESI-MSanalyses, a Waters XTerra® C18 5 μm column was used. Preparative highperformance liquid chromatography (HPLC) runs for syntheticintermediates utilized an Atlantis® Prep T3 OBD™ 5 μM column (eluents0.1% TFA (v/v) in water and MeCN; gradient: 0-1.5 min, 5-100% B; 1.5-2.0min 100% B). For radiolabeled compounds, preparative scale HPLCpurification was achieved using a Machery-Nagel Nucleodur C18 Pyramid250×10 mm Vario-Prep column (60:40 0.1% trifluoroacetic acid (v/v) inwater-acetonitrile (MeCN) at 5.5 mL·min⁻¹) with a 254 nm UV detector andradiodetector connected in series. Analytical HPLC of radiolabeledcompounds was performed employing a Grace VYDAC (218TP510) C18reversed-phase column (eluents 0.1% TFA (v/v) in water and MeCN;gradient: 0-17 min, 5-60% B; 17-21 min, 60-95% B; 21-24 min, 95% B;24-25 min, 95-5% B; 25-30 min, 5% B; 2 mL·min⁻¹) with a dual-wavelengthUV-vis detector and a flow-through gamma detector connected in series.HyperSep C18 cartridges were purchased from Thermo Electron (Bellefonte,Pa.) and Sep-pak VAC Alumina-N cartridges from Waters (Milford, Mass.).High-resolution electrospray ionization (ESI) mass spectra were obtainedon a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform massspectrometer (FT-ICR-MS) in the Department of Chemistry InstrumentationFacility at the Massachusetts Institute of Technology. IC₅₀ assays wereanalyzed using a Tecan (Männedorf, Switzerland) Safire2 microplatesystem. All kinetic data were analyzed using Prism 4 (GraphPad, LaJolla, Calif.) for Mac.

Preparation of (Z)-Cyclooct-4-enol (1)

9-Oxabicyclo[6.1.0]non-4-ene (4.2 g, 33.8 mmol) was added slowly toLiAlH₄ (1.2 g, 30.4 mmol) suspended in diethyl ether (100 mL). Afterstirring at room temperature for 4 hours, the reaction was worked up bythe sequential addition of 4 mL H₂O, 4 mL 25% NaOH(aq), and 4 mL of H₂O.The resulting mixture was filtered and the filtrate dried (Na₂SO₄) andfiltered again. The clear ether solution was concentrated to give 4.1 gof (Z)-cyclooct-4-enol in 96.1% yield.

Preparation of (Z)-2-(Cyclooct-4-enyloxy)acetic acid (2)

(Z)-Cyclooct-4-enol (2.0 g, 15.8 mmol) was added slowly to a suspensionof sodium hydride (1.3 g of 60% dispersion in mineral oil, 31.7 mmol) in50 mL THF. This was stirred at reflux for 1 hour, then a solution ofiodoacetic acid (2.9 g, 15.8 mmol) in 10 mL THF was added and reflux wascontinued for 4 hours, then the reaction was cooled to room temperatureand concentrated under reduced pressure. The residue was dissolved in10% NaOH(aq) (50 mL) and extracted with Et₂O (2×25 mL). The pH of theaqueous solution was lowered to (4) by the addition of 6N HCl andextracted again with DCM (2×25 mL). Separately, the organic solutionswere dried (MgSO₄), filtered, and concentrated by rotary evaporator. Theether extraction resulted in 1.0 g of starting (Z)-cyclooct-4-enol andthe DCM extraction resulted in 1.9 g of (Z)-2-cyclooct-4-enyloxy)aceticacid (2) (65.5% yield).

Preparation of (Z)-2-(Cyclooct-4-enyloxy)ethanol (3)

(Z)-2-Cyclooct-4-enyloxy)acetic acid (1.9 g, 10.3 mmol) was added to asuspension of LiAlH₄ (0.4 g, 9.5 mmol) in Et₂O (10 mL) at 0° C., warmedto room temperature, and was stirred for 24 hours. Unreacted LiAlH₄ wasquenched with 10% HCl(aq) and the reaction diluted with 30 mL H₂O. TheEt₂O layer was separated and the aqueous solution was extracted withEt₂O (2×10 mL). The combined Et₂O solutions were dried (MgSO₄),filtered, and concentrated. The crude mixture was subjected to columnchromatography (2:3 hexanes:ethyl acetate) to give 1.4 g of(Z)-2-(cyclooct-4-enyloxy)ethanol (3) (Rf=0.58), a 78.3% yield.

Preparation of (E)-2-(Cyclooct-4-enyloxy)ethanol (4)

(Z)-2-(Cyclooct-4-enyloxy)ethanol (1.0 g, 5.9 mmol) was converted to the(E)-isomers following a previously described cycle/trap method (M.Royzen et al., J. Am. Chem. Soc. 2008, 130, 3760-3761) with theexception of using methyl 4-(trifluoromethyl)benzoate (1.1 g, 7.9 mmol)as the photochemical sensitizer. The (E)-isomers were released from the10% AgNO₃ silica gel with 50 mL of 30% ammonium hydroxide(aq) and 50 mLDCM by stirring for 10 minutes. The suspension was filtered, theorganics separated, dried (MgSO₄) and concentrated to give 500 mg of apale yellow oil. This crude mixture was subjected to columnchromatography (2:1 pentane:Et₂O; starting material Rf=0.23) securing106 mg of the minor isomer (Rf=0.31) and 262 mg of the major isomer(Rf=0.14).

Preparation of (E)-2-(Cyclooct-4-enyloxy)ethyl 4-methylbenzenesulfonate(5)

The major isomer of (E)-2-(cyclooct-4-enyloxy)ethanol (180 mg, 1.1mmol), tosyl chloride (262 mg, 1.4 mmol), and triethylamine (214 mg, 2.1mmol) were combined in acetonitrile (6 mL). Reaction progress wasmonitored by TLC (1:1 hexanes:EA; starting material Rf=0.50 and desiredproduct Rf=0.84). After 2 hours stirring at room temperature, thereaction mixture was filtered and concentrated by rotary evaporation.The crude mixture was subjected to column chromatography (1:1hexanes:ethyl acetate) to give 298 mg of (E)-2-(cyclooct-4-enyloxy)ethyl4-methylbenzenesulfonate (5), a 84% yield.

Preparation of (E)-5-(2-Fluoroethoxy)cyclooct-1-ene (6^(19F))

(E)-2-(Cyclooct-4-enyloxy)ethyl 4-methylbenzenesulfonate (19 mg, 58.6μmol) diluted in THF (1 mL) was treated with a tetrabutylammoniumfluoride in THF (123 μL of 1 M solution). Reaction progress wasmonitored by TLC (2:1 pentane:Et20; starting material Rf=0.62 anddesired product Rf=0.92). After stirring for 2 hours, the mixture wasconcentrated and the resulting amber oil subjected to columnchromatography (silica gel, pentane) isolating 9.2 mg of(E)-5-(2-fluoroethoxy)cyclooct-1-ene (6¹⁹F (91.1%)).

Preparation of AZD2281-Tz (9)

Cyclohexylcarbodiimide polystyrene resin (127 mg, 2.3 mmol/g) was addedto a solution of 4-[[4-Fluoro-3-(4-(5-oxopentanamide)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one 7 (70 mg, 0.15mmol) in dichloromethane (10 mL) and the resulting mixture was stirredgently for 7 hours at room temperature. Subsequently, tetrazine amine(8) (55 mg, 0.29 mmol) and triethylamine (81 μL, 0.58 mmol) was addedand the mixture stirred for another 60 minutes, before the reactionmixture was filtered and volatiles removed in vacuo. The crude materialwas purified via HPLC, yielding the title compound as a pink solid (24mg, 37 25%).

Preparation of 1-AZD2281-¹⁹F (10^(19F))

A solution of AZD2281-Tz (9) in DMSO (10 μL, 1 mM, 0.01 μmol) was addedto (6¹⁹F) (10 uL, 1 mM in DMSO, 0.01 μmol) and agitated for 30 minutes,before the crude reaction mixture was purified via HPLC-chromatography.HRMS-ESI [M+H]+m/z calcd. for [C44H47F2N7O5]+792.368, found 792.3690.

Preparation of 1-AZD2281-¹⁶O (10^(16O))

A solution of AZD2281-Tz (9) in DMSO (10 μL, 1 mM, 0.01 μmol) was addedto 4 (10 uL, 1 mM in DMSO, 0.01 μmol) and agitated for 30 minutes,before the crude reaction mixture was purified via HPLC-chromatography.HRMS-ESI [M+H]+m/z calcd. for [C44H48FN7O6]+ 790.3723, found 790.3707.

Preparation of 1-AZD2281-¹⁸O (10^(18O))

A solution of HPLC purified 1-AZD2281-¹⁸F (10^(18F)) was allowed tostand at room temperature for 48 h to allow all radioactive ¹⁸F todecay. HRMS-ESI [M+H]+m/z calcd. for [C44H48FN7O518O]+ 792.3765, found792.3753.

Preparation of4-[[4-Fluoro-3-((6-hydroxyhexanoyl)piperazine-1-carbonyl)phenyl]methyl]2H-phthalazin-1-one(12)

4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(11) (250 mg, 0.68 mmol), HBTU (337 mg, 0.89 mmol) and triethylamine(285 μL, 1.18 mmol) were added to a solution of 6-hydroxyhexanoic acid(180 mg, 1.36 mmol) in DMF (3.0 mL) and the reaction mixture was stirredat room temperature for 60 minutes, before dichloromethane (8 mL) andwater (8 mL) were added, the organic phase separated and washed withwater (3×8 mL). The organic phase was dried over MgSO₄, volatilesremoved in vacuo and the resulting crude material purified via HPLC,yielding the title compound as a clear solid (55.6 mg, 0.12 mmol, 17%).

Preparation of4-[[4-Fluoro-3-((6-tosylhexanoyl)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(13)

Triethylamine (29 μL, 0.21 mmol) was added to a solution ofp-toluenesulfonyl chloride (20 mg, 0.10 mmol) and4-[[4-Fluoro-3-((6-hydroxyhexanoyl)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (12) (25 mg, 0.052 mmol) indichloromethane (5 mL), the reaction mixture was stirred at roomtemperature overnight and purified via HPLC, yielding the title compoundas a clear solid (7.8 mg, 0.01 mmol. 24%).

Preparation of4-[[4-Fluoro-3-((2-hydroxyacetyl)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(15)

4-[[4-Fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(11) (86 mg, 0.24 mmol), HBTU (116 mg, 0.30 mmol) and triethylamine (164μL, 1.18 mmol) were added to a solution of 2-hydroxyacetic acid (36 mg,0.48 mmol) in DMF (1.5 mL) and the reaction mixture was stirred at roomtemperature for 40 minutes, before dichloromethane (4 mL) and water (4mL) were added, the organic phase separated and washed with NaOH (0.2 M,3×4 mL) and water (3×4 mL). The organic phase was dried over MgSO₄,volatiles were removed in vacuo and the resulting crude material waspurified via HPLC, yielding the title compound as a clear solid (24.3mg, 0.06 μmol, 50%).

Preparation of4-[[4-Fluoro-3-((2-tosyl-acetyl)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(16)

Triethylamine (53 μL, 0.38 mmol) was added to a solution ofp-toluenesulfonyl chloride (36 mg, 0.19 mmol) and4-[[4-Fluoro-3-((2-hydroxyacetic acid)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one (15) (40 mg, 0.094 mmol) in dichloromethane(5 mL), the reaction mixture was stirred at room temperature overnightand purified via HPLC, yielding the title compound as a clear solid (34mg, 0.06 mmol. 32%).

Preparation of 2-AZD2281-¹⁹F (17^(19F)).

Freshly dried NaF (8.4 mg, 0.2 mmol) was added to a solution of4-[[4-fluoro-3-((2-tosyl-acetyl)piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(16) (10 mg, 0.02 mmol) in dry acetonitrile (2 mL) and was stirred for 6hours at 40° C. before the reaction mixture was purified via HPLC,yielding the title compound as a clear solid (1.2 mg, 2.8 μmol, 16%).

B. Radiochemistry4-(4-fluoro-3-(4-(2-18F-fluoroacetyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one(17^(18F))

[¹⁸F]-F⁻, n.c.a. (no carrier added), (˜77 MBq, 2.4±0.9 mCi) in H₂ ¹⁸O(˜150 μL), 250 μL of a 75 mM tetrabutylammonium bicarbonate(^(n)Bu₄NHCO₃) solution in water, and 750 μL of MeCN were combined in a10-mL test tube and heated (microwave) to 98° C. under a stream ofargon. At 4, 8 and 12 minutes, 1 mL of MeCN was added and evaporatedoff. To the dried [¹⁸F]-F⁻ (n.c.a.)/^(n)Bu₄NHCO₃ was added 100 μL of a35 mM solution of tosylate (16) in dimethylformamide and heated to 40°C. for 10 minutes. To remove unreacted [¹⁸F]-fluoride, this mixture wasfiltered through an Alumina-N cartridge (100 mg, 1 mL, Waters) to give16 μCi in the filtrate. HPLC coinjection of a sample of (17^(19F)) withan aliquot of this filtrate demonstrated formation of the desiredproduct (17^(18F)) in 30 minutes and 0.8% dcRCY.

2-¹⁸F-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (6^(18F))

2-¹⁸F-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (¹⁸F-TCO) was prepared usingSynthra RN Plus automated synthesizer (Synthra GmbH, Hamburg, Germany)operated by SynthraView software in an average time of 40 minutes. Thetarget well was charged with [¹⁸F]-F⁻, n.c.a., (˜1110 MBq, 30±10 mCi) inH₂ ¹⁸O (150 μL), 250 μL of a 75 mM tetrabutylammonium bicarbonate (TBAB)solution in water, and 200 μL of MeCN. The synthesizer reagent vialswere filled as follows: A2 with MeCN (350 μL), A3 with tosylate (5) (4.0mg, 12.3 μmol) in DMSO (400 μL), A5 with DMSO (50 μL), and B2 with H₂O(800 μL). The [¹⁸F]-F-/TBAB solution was transferred to Reaction Vessel#1 and dried by azeotropic distillation of the acetonitrile/watersolution by heating to 60° C. under reduced pressure and a flow of argonto achieve ˜310 mbar for 2 minutes followed by 98° C. and 270 mbar for 4minutes. Reaction Vessel #1 was cooled to 50° C., tosylate (5) in DMSO(400 μL) added, the reaction vessel pressurized to 2000 mbar, and heatedto 90° C. for 10 minutes. Cooled to 30° C., the mixture was filteredthrough an Alumina-N cartridge (100 mg, 1 mL, Waters) into ReactionVessel #2. The Alumina-N cartridge was washed with DMSO (50 μL) and thecombined filtrates were diluted with water (800 μL). This solution wassubjected to preparative HPLC purification. (61⁸¹) was collected(tR=10.1 min) in 4-5 mL of solvent, isolated by C18 solid phaseextraction and eluted with DCM (600 μL) to give 7.7±3.4 mCi of 618F in44.7±7.8% (n=16) decay-corrected radiochemical yield (dcRCY) in anaverage time of 41 min from the end of drying of [¹⁸F]-F⁻ (n.c.a.).Analytical HPLC demonstrated >93% radiochemical purity of (6^(18F)).

1-AZD2281-¹⁸F (10^(18F))

To the above described (6¹⁸F)/DCM solution was added AZD2281-Tz (9) (7μL of 18.5 mM DMSO solution, 0.13 μmol) and stirred at rt for 3 minutes.The mixture was concentrated with a gentle stream of argon,reconstituted in 1:1 MeCN/H₂O (to a volume of 1.3 mL), subjected topreparative HPLC purification (tR=6.0 min) and isolated by C18 solidphase extraction. Elution with MeOH (600 uL) followed by evaporation ofsolvent provided 2.3±0.8 mCi (n=3) of (10^(18F)).

1-AZD2281-¹⁸O (10^(18O))

A solution of HPLC purified 1-AZD2281-^(18F) (10^(18F)) was allowed tostand at room temperature for 48 hours to allow all radioactivity todecay. HRMS-ESI [M+H]+m/z calcd. for [C44H48FN7O518O]+ 792.3765, found792.3753.

C. Parp1 Ic₅₀ Determination

To assess the effect of the TCO/Tz ligand on target affinity, acolorimetric assay was employed to measure PARP1 activity.

A commercially available colorimetric assay (Trevigen, Gaithersburg,Md.) was used to measure PARP activity in vitro in the presence ofinhibitors. Tenfold dilutions of compounds (6^(19F)), (10^(19F)),(17^(19F)) (final concentration 4 μM to 0.04 nM) and (9) (1 μM to 0.1nM) were incubated with 0.5 units PARP HSA for 10 minutes inhistone-coated 96-well plates. All experiments were carried out intriplicate. Control samples did not contain inhibitor and backgroundmeasurement samples did not contain PARP1. All reaction mixtures wereadjusted to a final volume of 50 μL and a maximum final concentration0.4% DMSO in assay buffer. The remainder of the assay was performedaccording to the manufacturer's instructions. PARP activity was measuredby absorbance at 450 nm in each well using a Safire2 microplate reader.IC₅₀ values were calculated using the Prism software package.

The published value for AZD2281 is 5 nm (K. A. Menear et al., J. Med.Chem. 2008, 51, 6581-6591; and D. V. Ferraris, J. Med. Chem. 2010, 53,4561-4584), identical to what we observed in our assay. Conventionallyfluorinated (17¹⁹F) had an IC₅₀ value of 5.2±1.1 nm, consistent with thesmall side group. Compound (9) showed an IC₅₀ value of 8.4±1.3 nm, quiteremarkable given the bulkier side chain. Cycloaddition fluorinated(10^(19F)) had an IC₅₀ value of 17.9±1.1 nm (FIG. 4), still in the lowdouble-digit nanomolar range and likely sufficient for imaging purposes.These findings are also in agreement with previous results showing thatmodification of AZD2281 at the piperizine position only minimallyperturbs the ability to bind PARP1 (T. Reiner, S. Earley, A. Turetsky,R. Weissleder, ChemBioChem 2010, 11, 2374-2377).

Example 3 A. Synthesis Materials and Methods.

Unless otherwise noted, all reagents were purchased from Sigma-Aldrich(St. Louis, Mo.) and used without further purification.Cyclohexylcarbodiimide polystyrene resin was purchased from EMDbiosciences (Gibbstown, N.J.). Magnetic amine-decorated beads werepurchased from Invitrogen (Dynabeads® M270 amine, Carlsbad, Calif.).Oregon Green-Tz[1],4-[[4-fluoro-3-(piperazine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one(AZD2281, (4)), AZD2281-Tz (5) and (E)-2-(Cyclooct-4-enyloxy)ethyl4-methylbenzenesulfonate (2) were synthesized as described earlier (K.A. Menear et al., J. Med. Chem. 2008, 51, 6581-6591; and E. J. Keliheret al., ChemMedChem 2010, DOI: 10.1002/cmdc.201000426). ¹⁸F-Fluoride(n.c.a.) in ¹⁸O-enriched water was purchased from PETNET (Woburn,Mass.). Automated synthesis of ¹⁸F-labeled trans-cyclooctene was carriedout using a Synthra RN Plus automated synthesizer (Synthra GmbH,Hamburg, Germany) operated by SynthraView software. For non-radioactivecompounds, LC-ESI-MS analysis and HPLC-purifications were performed on aWaters (Milford, Mass.) LC-MS system. For LC-ESI-MS analyses, a WatersXTerra® C18 5 μm column was used. Preparative high performance liquidchromatography (HPLC) runs for synthetic intermediates utilized anAtlantis® Prep T3 OBD™ 5 μM column (eluents 0.1% TFA (v/v) in water andMeCN; gradient: 0-1.5 min, 5-100% B; 1.5-2.0 min 100% B). Forradiolabeled compounds, preparative scale HPLC purification was achievedusing a Machery-Nagel Nucleodur C18 Pyramid 250×10 mm Vario-Prep column(60:40 0.1% trifluoroacetic acid (v/v) in water-acetonitrile (MeCN) at5.5 mL·min⁻¹) with a 254 nm UV detector and radiodetector connected inseries. Analytical HPLC of radiolabeled compounds was performedemploying a Grace VYDAC (218TP510) C18 reversed-phase column (eluents0.1% TFA (v/v) in water and MeCN; gradient: 0-17 min, 5-60% B; 17-21min, 60-95% B; 21-24 min, 95% B; 24-25 min, 95-5% B; 25-30 min, 5% B; 2mL·min-1) with a dual-wavelength UV-vis detector and a flow-throughgamma detector connected in series. HyperSep C18 cartridges werepurchased from Thermo Electron (Bellefonte, Pa.) and Sep-pak VACAlumina-N cartridges from Waters (Milford, Mass.). High-resolutionelectrospray ionization (ESI) mass spectra were obtained on a BrukerDaltonics APEXIV 4.7 Tesla Fourier Transform mass spectrometer(FT-ICR-MS) in the Department of Chemistry Instrumentation Facility atthe Massachusetts Institute of Technology. IC₅₀ assays were analyzedusing a Tecan (Männedorf, Switzerland) Safire2 microplate system. Allkinetic data were analyzed using Prism 4 (GraphPad, La Jolla, Calif.)for Mac.

Preparation of Magnetic Trans-Cyclooctene Scabenger Resin.

One problem during the synthesis of ¹⁸F-radiolabeled compounds is thatin most cases large amounts of precursors are used to efficiently reactwith small quantities of ¹⁸F. The resulting mixtures are typicallypurified by HPLC to remove the excess starting material, which in mostcases will compete with the radiolabeled probe for the targeted bindingsites. To avoid lengthy HPLC purifications, we designed atrans-cyclooctene resin to “pull out” excess tetrazine-conjugatedAZD2281 derivatives from the reaction mixture (FIG. 5A).

Magnetic amine-decorated beads (400 μL, approx. 12 mg) were separatedfrom the storage buffer and washed with a mixture of PBS 1× and DMF(3×400 μL, PBS 1×/DMF=1/1) and were resuspended in PBS 1×/DMF (400 μL,1/1). Trans-Cyclooctene 1 was added (40 μL, 75 mM in DMF) and themixture stirred gently for 2.5 h. Subsequently, the beads were washedwith a mixture of PBS 1× and DMF (4×400 μL, PBS 1×/DMF=1/1), resuspendedin PBS 1×/DMF (400 μL, 1/1) and stored at 4° C. (FIG. 5B).

Preparation of 2-[¹⁸F]-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (3)

(2-[¹⁸F]-1-ene (3) was synthesized similar to methods described earlier(E. J. Keliher et al., ChemMedChem 2010, DOI: 10.1002/cmdc.201000426;FIG. 5C). Briefly, 2-[¹⁸F]-(E)-5-(2-fluoroethoxy)cyclooct-1-ene(′⁸F-TCO) was prepared using Synthra RN Plus automated synthesizer(Synthra GmbH, Hamburg, Germany) operated by SynthraView software in anaverage time of 40 minutes. The target well was charged with [¹⁸F]-F-,n.c.a., (˜1110 MBq, 30±10 mCi) in H₂ ¹⁸O (150 μL), 250 μL of a 75 mMtetrabutylammonium bicarbonate (TBAB) solution in water, and 200 μL ofMeCN. The synthesizer reagent vials were filled as follows: A2 with MeCN(350 μL), A3 with tosylate 2 (4.0 mg, 12.3 μmol) in DMSO (400 μL), A5with DMSO (50 μL), and B2 with H₂O (800 μL). The [¹⁸F]-F-/TBAB solutionwas transferred to Reaction Vessel #1 and dried by azeotropicdistillation of the acetonitrile/water solution by heating to 60° C.under reduced pressure and a flow of argon to achieve ˜310 mbar for 2minutes followed by 98° C. and 270 mbar for 4 minutes. Reaction Vessel#1 was cooled to 50° C., tosylate (2) in DMSO (400 μL) added, thereaction vessel pressurized to 2000 mbar, and heated to 90° C. for 10minutes. Cooled to 30° C., the mixture was filtered through an Alumina-Ncartridge (100 mg, 1 mL, Waters) into Reaction Vessel #2. The Alumina-Ncartridge was washed with DMSO (50 μL) and the combined filtrates werediluted with water (800 μL). This solution was subjected to preparativeHPLC purification. ¹⁸F-TCO (3) was collected (tR=10.1 min) in 4-5 mL ofsolvent, isolated by C18 solid phase extraction and eluted with DCM (600μL), concentrated with a gentle stream of argon, reconstituted in 1:1MeCN/H₂O (to a volume of 1.0 mL) to give 6.7±2.2 mCi of (3¹⁸) in37.0±1.9% (n=4) decay-corrected radiochemical yield (dcRCY) in anaverage time of 41 minutes from the end of drying of [¹⁸F]-F⁻ (n.c.a.).Analytical HPLC demonstrated >93% radiochemical purity (RCP) of(3^(18F)).

Preparation of 18F-AZD2281 (6)

¹⁸F-AZD2281 (6) was synthesized similar to methods described earlier (E.J. Keliher et al., ChemMedChem 2010, DOI: 10.1002/cmdc.201000426).AZD2281-Tz (5) (7 μL of an 18.5 mM DMSO solution (0.13 μmol)) was addedto a solution of (3^(18F)) (2.3 mCi) and stirred at room temperature for3 minutes. This results in quantitative conversion of ¹⁸F-TCO (3),yielding a mixture of ¹⁸F-AZD2281 (6) and excess AZD2281-Tz (5; FIG.5E). The solution was treated with 4 mole equivalents of TCO-beads (0.52μmol TCO), vortexed and allowed to stand for 5 minutes. The beads wereremoved by magnetic separation and the reaction mixture analyzed byHPLC, 92.1±0.4% dcRCY and 96.0±2.0% (n=3) RC purity. Treatment of thissolution for 5 minutes with the TCO-decorated scavenger resin (4 molequiv of TCO) removed unreacted AZD2281-Tz with minimal loss(approximately 4%) of radiolabeled compound (6). Magnetic removal of thebeads provided (6) in (92.1 0.4)% dcRCY, which was then reconstituted ina medium suitable for animal injection (FIG. 5A). HPLC analysis of thereaction mixture before and after treatment with the TCO-decoratedscavenger resin shows that the absorption peak resulting from AZD2281-Tzcompletely vanishes, whereas the activity peak resulting from¹⁸F-AZD2281 persists (FIG. 5F).

B. Loading of Magnetic TCO Scavenger-Resin

Loading of the magnetic TCO-scavenger resin was determined by separatingboth the beads and their unmodified precursors (each 50 μL, approx. 1.5mg) from the storage solution (PBS 1×/DMF=1/1) and storage-buffer,respectively and washing with a mixture of PBS 1× and DMF (3×50 μL, PBS1×/DMF=1/1). They were resuspended in a solution of Oregon Green-Tz (500μM, 50 μL; PBS 1×/DMF=1/1), and stirred gently for 50 min. The amount ofOregon Green-Tz “pulled out” of the solution by trans-cyclooctene wasmeasured by determining the solution's absorption at 504 nm and theloading (specific-non-specific adsorption) calculated to be 13 μmol/g.

C. Assays Cell Culture

MDA-MB-231 and MDA-MB-436 cells were obtained from ATCC (Manassas, Va.)and cultured in RPMI 1640 supplemented with 10% fetal bovine serum,L-glutamine, and penicillin/streptomycin (each Invitrogen, Carlsbad,Calif.) at 37° C. and 5% CO₂.

PARP1 Expression Analysis

PARP1 expression in MDA-MB-231 and MDA-MB-436 cells was determined byimmunoblot analysis. Cells were lysed in RIPA buffer (Cell SignalingTechnology, Danvers, Mass.) containing complete protease inhibitors(Roche Applied Science, Indianapolis, Ind.), and protein concentrationwas quantified using the microBCA assay (Pierce, Rockford, Ill.).Lysates containing SDS loading buffer were boiled for 10 minutes,proteins were separated by SDS-PAGE, then transferred to PVDF membranesusing the iBlot (Invitrogen, Carlsbad, Calif.). Membranes were blockedovernight at 4° C. using 5% milk (for PARP1 blot) and 5% BSA (for GAPDHblot) in TBSTween 20, probed with antibodies against PARP1 (XY-7, SantaCruz Biotechnologies, Santa Cruz, Calif.) and GAPDH (Millipore,Temecula, Calif.) for 1 hour at room temperature, then washed with PBS.Membranes were then incubated with appropriate secondary antibodiesconjugated to HRP for 1 hour at room temperature. After washing,membranes were incubated with SuperSignal West Pico chemiluminescentsubstrate (Pierce, Rockford, Ill.) for 5 minutes, and exposed to film.

The IC₅₀ value of ¹⁸F-AZD2281 against PARP1 was determined to be (17.91.1) nm in biochemical assays using the isolated enzyme (AZD2281 itselfhas an IC₅₀ value of 5 nM). This demonstrates PARP1 to be an idealtarget for the rapid screening of ¹⁸F-labeled inhibitors, as it canaccommodate relatively large prosthetic groups that do not affectbinding affinity.

Cell Based Assays

For cell based assays, MDA-MB-231 and MDA-MB-436 cells (50,000cells/well) were seeded into 96-well plates and allowed to attach overnight at 37° C. and 5% CO₂. Ten-fold dilutions of AZD2281 4 (finalconcentration: 10 μM to 0.01 nM) were added to the wells and incubatedfor 10 minutes, before 5 μCi of 18F-AZD2281 6 (5 μL, 0.2 μC/μL) wereadded and the cells incubated for 20 minutes. Then, the medium wasreplaced and waited for another 10 minutes, before the cells were washedwith PBS (3×100 μL) and subjected to gamma-counting.

Cellular uptake of ¹⁸F-AZD2281 was determined by quantification of theremaining activity after incubation and washing of the adherent cells.¹⁸F-AZD2281 was shown to be cell-permeable and its uptake inhibitableupon addition of excess nonradioactive AZD2281 (FIG. 6). Uptake of¹⁸F-AZD2281 was lower for MDA-MB-231 cells than for MDA-MB-436 cells,which correlates with protein expression of PARP1 in the respective celllines.

Mice

Non-tumor bearing Nu/Nu mice were injected with (6^(18F)) and used fororgan distribution immediately after injection over a time period of 2hours. Tumor-bearing mice received s.c. injections with 5×106 MDA-MB-436cells (American Type Culture Collection) in Matrigel (BD Biosciences)into each flank and underwent imaging 7 days later (injection of(6^(18F)) and immediate imaging over a time period of 2 hours). All micewere anesthetized (isoflurane 1.5%; O₂ 2 L/min) during imaging with agas delivery system integrated into a multimodal imaging cartridge. Allanimal experiments were approved by Massachusetts General Hospital'sInstitutional Review Committee.

Images were acquired on the Siemens Inveon PET-CT. Each PET acquisitionwas 120 minutes in duration. PET was reconstructed from 600 millioncoincidental 511 keV photon counts on a series of LSO (lutetiumoxyorthosilicate) scintillating crystals. Counts were rebinned in 3D byregistering photons spanning no more than 3 consecutive rings andreconstructed into sinograms using a high resolution Fourier Rebinalgorithm. Sinograms also yielded a 3D mapping of positron signal usinga 2D filtered back-projection algorithm and a Ramp filter with a Nyquistcut-off of 0.5. Datasets were anisotropic matrices containing128×128×159 pixels, measuring 0.796 mm/pixel in the z direction and0.861 mm/pixel in the x and y directions. Calibration of PET signalpreceded all scans by scanning an 8.0 cm in diameter cylindrical phantomcontaining a known amount of ¹⁸F isotope. Data are expressed as standarduptake values (SUV), which normalizes activity measurements to bodyweight and injected activity.

For anatomic reference of PET signal, x-rays were projected over 360degrees to create a computed tomographic (CT) image. X-ray projectionshad a cone beam angle of 9.3 degrees and a power of 80 keV. A 500 μAanode source was located 347 mm from the center of rotation and x-rayswere incident on a CCD detector containing 2048 transaxial and 3072axial pixels. Projections were calibrated using 70 dark and 70 lightimages, interpolated bilinearly, processed through a Shepp-Logan filter,and then reconstructed using a filtered back projection algorithm. CTdatasets were isotropic matrices with a total of 512×512×768 pixelsmeasuring 110 μm³. During CT acquisition, iodine contrast was infusedinto the tail vein at a rate of 35 μL/min to enhance intravascularcontrast. Projections were acquired at end expiration of the respiratorycycle using a BioVet gating system (M2M Imaging, Cleveland, Ohio) andtotal CT acquisition time was ˜10 minutes. PET-CT fusion and imageanalysis were performed using Inveon Research Workplace 3.0 (Siemens).Three-dimensional visualizations were produced with the DICOM viewerOsiriX (The OsiriX foundation, Geneva, Switzerland).

FIG. 7A displays the distribution of the PET probe in the Nu/nu mice at10, 30, and 50 minutes. The images clearly show initial localization ofthe probe to be mainly in liver, gall bladder, and intestines,consistent with hepatobiliary excretion. After 50 minutes (FIG. 7A), themajority of the probe had left the bloodstream (t_(1/2)=6 min) and wasexcreted through the large intestines. FIG. 7B shows a three-dimensionalreconstruction of a tumor-bearing mouse injected with 30 mCi of¹⁸F-AZD2281. Uptake in the tumors is clearly visible. Immediately afterimaging, the mice were injected with 1 mg AZD2281 BID. Re-injection of30 mCi of ¹⁸F-AZD2281 confirmed inhibition of the probe s uptake intothe tumors (FIGS. 7B and 7C). Therefore, owing to the rapid decay rateof ¹⁸F, each mouse serves as its own control on subsequent days ofimaging, facilitating direct comparisons of standardized uptake value(SUVs).

Example 4. Non-Invasive Monitoring of Treatment Response UsingAZD2281-¹⁸F

¹⁸F-AZD2281 was synthesized as described herein. Nu/Nu mice bearingeither A2780 tumors (treatment response) or A2780, Paca2, Panc1 andSKOV3 (differential uptake) were injected with the hot probe (500-750μCi) and imaged ˜1.5 hours thereafter. Treatment consisted of injectionof 0.5-1.0 mg AZD2281 (Olaparib) 18 hours before imaging (ip,interperitoneal) or −1.5-3.5 hours before imaging (iv, intravenous).Uptake was determined non-invasively by drawing SUVs (Standard UptakeValues).

FIG. 8A shows that FDG, the current gold standard in cancer imaging, isnot able (based on tumor/muscle signal intensity) to distinguish betweentreated untreated tumors. Injection of Olaparib (AZD2281; Tx=treatment,starting between day 0 and day 1) into tumors lets the signal of¹⁸F-AZD2281 drop, which is a major advantage in terms of predicting if ahuman tumor will respond to treatment with Olaparib or other PARPinhibitors. FIG. 8B shows the relative uptake of ¹⁸F-AZD2281 activity ofdifferent ovarian and pancreatic tumors (A2780 and SKOV3=ovarian, PANC1and PACA2=pancreatic). This data indicates that a wide variety of tumorsrespond to ¹⁸F-AZD2281 treatment, and that those cancers can be detectedusing the compounds provided herein.

Example 5. Comparison of Uptake and Expression

RAW 264.7, Panc-1, MIA Paca2, A2780, OVCAR429, UCI 101, UCI 107, SKOV-3,OVCAR-3, and OV-90 cells (200 μL, 35,000 cells/mL) were each seeded intheir respective growth medium on 96-well plates and allowed to attachfor 48 hours. They were incubated with AZD2281-BODIPY FL (2 μL, 100 μL)for 20 minutes (37° C.) before the medium was removed and cells werewashed (1×, medium, 200 μL). Cells were then fixed with paraformaldehyde(4% in PBS) and washed with ice cold PBS/0.1% Triton X-100/0.5% bovineserum albumin (3×200 μL, 3rd wash left for 30 min). Cells were incubatedwith anti-PARP-1/2 Rab (1:50; SCBT, Santa Cruz, Calif.) at 4° C.overnight. Cells were washed with PBS/0.1% Triton X-100 (3×200 μL)before stained with secondary IgG-Cy5 Gab (1:100; Millipore, Billerica,Mass.) for 3 h at 4° C. Cells were then washed with PBS (1×, 200 μL),stained with Hoechst (Invitrogen, Carlsbad, Calif.) and blue whole cellstain (Thermo Scientific, Waltham, Mass.) for 30 minutes at roomtemperature, and washed with PBS (3×, 200 μL) before imaging andquantification of the respective fluorescence signals.

FIG. 9A shows that AZD2281-BODIPY FL (green—inner halo) localizes withthe nucleus (as confirmed with the red signal (inner most signal),Chromatin (essentially a DNA stain)). This is where PARP1, the target,is located. It does not go into the perinuclear region (the rest of thecell), as shown by the blue stain, which basically colors the cellwalls. The composite combines all colors. FIG. 9B plots the amount offluorescence present in cells using AZD2281 and a PARP1/PARP2 antibody.The more PARP1/PARP2 there is in a cell line, the more AZD2281-BODIPY FLis taken up by the cell. FIG. 9C shows the same data as FIG. 9B, butplots the data differently. This figure shows that the R2 (a measure forcorrelation) is very high, indicating that AZD2281-BODIPY FL not onlytargets PARP1, but targets it quantitatively, allowing for an accuratemeasurement of the amount of PARP1 in a given cell line (or, in aclinical setting, tumor environment).

Example 6. Life Cell Imaging

For in vitro kinetic studies, HT1080 cells were labeled with a 1 μMconcentration of AZD2281 fluorophore conjugate in cell culture mediumand imaged on an inverted Deltavision epifluorescence microscope using a37° C. heated stage and 5% CO₂ atmosphere for various times. Aftersubtracting the background fluorescence, the data were fitted to anexponential association curve. For clearance, cells were labeled at 1 μMfor 1 hour until the cells had reached equilibrium. Cells were washedtwice in cell culture media and imaged continuously (BODIPYFL conjugate)or after various time points (BODIPY578 and BODIPY650). The loss influorescence was fitted to a biexponential decay curve.

In FIGS. 10 and 11, three different AZD2281 fluorophores were testedusing life cell imaging. The cells are not fixed, but in their mostnatural state (as close as possible to using intravital imaging). First,the compounds were added to the medium surrounding the cells, thecompounds entered the cells, and when the medium (which containedAZD2281-fluorophores) was replaced, the AZD2281-fluorophores were washedout of the perinuclear region (where PARP1 is not located). However, thecompounds were retained in the nuclear region (where PARP1 is located),showing that all the compounds tested work in an in vitro setting. Table2 details the properties of the compounds tested, obtained by serialimaging techniques.

TABLE 2 AZD2281 Uptake Clearance (weighted) Properties BODIPYFLPerinuclear = 1.2 min Perinuclear = 1.7 min MW = 640 Nucleus = 1.1 minNucleus = slow (remeasure) BODIPY578 Perinuclear = 5.4 min Perinuclear =6.9 min MW = 677 Nucleus = 3.6 min Nucleus = 23.8 min BODIPY650Perinuclear = 21.2 min Perinuclear = 12.8 min MW = 895 Da Nucleus = 8.3min Nucleus = slow (remeasure)

Example 7. Cellular Resolution of Fluorescent AZD2281 Derivatives

For the in vivo studies, mice with window chambers were implanted with 1million HT-1080 cells expressing H2B-apple fluorescence protein. 75 nmolof AZD2281-BODIPYFL were dissolved in 75 μL of 1:1::DMAC:solutol and 75μL of PBS and injected via tail vein. Images of the vessels were takenusing a 20× water immersion objective on a laser scanning confocalmicroscope while the mouse was anesthetized with isoflurane. After 1hour, the non-specific uptake of the probe cleared revealing nuclearuptake of the probe.

FIG. 12 shows the behavior of AZD281-BODIPYFL in vivo. The compound wasinjected into a mouse by iv (10 seconds). The compound is first observedin the vascular system (where it was injected) and then concentrates ineach and every tumor cell of the tumor (red dots). This data shows theability of the compound to bind to PARP1 in vivo.

Example 8. Selective Tumor-Uptake of Fluorescent AZD2281 Derivatives

For ¹⁸F-AZD2281 standardized uptake values, five Nu/Nu mice eachreceived four subcutaneous injections containing SKOV-3, MIA Paca2,A2780, or Panc-1 cells in the flanks and shoulders (2.5×10⁶ cells in 100μL 70:30 PBS/BD matrigel per injection) (BD Biosciences, Bedford,Mass.). Tumors were allowed to grow for two weeks before imaging. 75nmol of AZD2281-BODIPYFL were dissolved in 75 μL of 1:1::DMAC:solutoland 75 μL of PBS and injected via tail vein. The agent was allowed tocirculate and clear for 45 minutes, before the mice were sacrificed,tumors and muscle tissue excised and the relative fluorescence uptake oftissues quantified against mice which were injected with just1:1::DMAC:solutol.

FIG. 13 shows that the uptake of AZD2281-BODIPY FL is not just specificon a cellular level, but also globally. Tumors have higher PARP1 levelsthan other tissue, and this is shown by the fact that very little of thecompound gets taken up by muscle cells compared to the tumor tissues.This data corresponds well to the in vitro data described above.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A compound of Formula (I):P-L_(n)-T_(m)-D or a pharmaceutically acceptable salt thereof, wherein:P is a PARP1 inhibitor; L is a linker; T is:

D is a detectable agent; m is 0 or 1; and n is 0 or
 1. 2. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein P isselected from the group consisting of: benzamide, quinolone,dihydroisoquinolinone, isoquinolinone, isoquinolone, benzopyrone, cyclicbenzamide, benzimidazole, indole, isoindolinone, nicotinamide, 3-AB,phthalazinone, and quinazolinone.
 3. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein P is selected from thegroup consisting of AZD2281, AG014699, ABT888, BSI201, BSI101, DR2313,FR 247304, GPI15427, GPI16539, MK4827, NU1025, NU1064, NU1085, PD128763,PARP Inhibitor II, PARP Inhibitor III, PARP Inhibitor VIII, PARPInhibitor IX, and TIQ-A.
 4. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein P is AZD2281.
 5. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein the compound of Formula (I) is a compound of Formula (II):

or a pharmaceutically acceptable salt form thereof, wherein: L is alinker; T is:

D is a detectable agent; m is 0 or 1; and n is 0 or
 1. 6. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein Dcomprises one or more of organic small molecules, inorganic compounds,nanoparticles, enzymes or enzyme substrates, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,and contrast agents.
 7. The compound of claim 6, or a pharmaceuticallyacceptable salt thereof, wherein D comprises a radioactive material, afluorescent material, or a mixture thereof.
 8. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein the compound ofFormula (I) is selected from the group consisting of:

wherein R is Texas Red, a cyanine dye, Alexafluor-680, a BODIPY dye, axanthene derivatives, a naphthalene dye, a courmarin derivative, anoxadiazole derivative, a pyrene derivative, an oxazine derivative, anacridine derivative, an arylmethine derivative, and a tetrapyrrolederivative.
 9. The compound of claim 8, or a pharmaceutically acceptablesalt thereof, wherein the compound of Formula (I) is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.
 10. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein the compoundof Formula (I) is:

or a pharmaceutically acceptable salt thereof.
 11. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier ordiluent.
 12. A method for detecting a cancer in a subject, the methodcomprising: (a) administering to a subject an effective amount of acompound of claim 1, or a pharmaceutically acceptable salt form thereof,and (b) detecting the detectable agent in the subject.
 13. The method ofclaim 12, wherein detecting the detectable agent comprises usinghistochemistry, fluorescence detection, chemiluminescence detection,bioluminescence detection, magnetic resonance imaging, nuclear magneticresonance imaging, positron emission tomography, single-photon emissioncomputed tomography, X-ray imaging, X-ray computed tomography,ultrasound imaging, or photoacoustic imaging.
 14. The method of claim12, wherein the cancer is selected from the group consisting ofpancreatic cancer, ovarian cancer, and breast cancer.
 15. The method ofclaim 12, wherein the compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, is administered before and after surgicalremoval of the cancer.
 16. A method for imaging a cancer cell, themethod comprising: (a) contacting a cancer cell with an effective amountof a compound of claim 1, or a pharmaceutically acceptable salt formthereof, and (b) imaging the cell.
 17. A method for monitoring thecancer treatment of a patient, the method comprising: (a) administeringto the patient, prior to a treatment, an effective amount of a compoundof claim 1, or a pharmaceutically acceptable salt form thereof, andimaging the patient; (b) administering to the patient, at a pointfollowing treatment, an effective amount the compound, or apharmaceutically acceptable salt thereof, and imaging the patient; and(c) comparing the image collected in step (a) with the image collectedin step (b) to monitor the treatment.
 18. The method of claim 17,wherein the treatment further comprises administration of an anti-canceragent.
 19. The method of claim 17, wherein the method further comprises:administering to the patient an effective amount of the compound, or apharmaceutically acceptable salt thereof, during treatment and imagingthe patient.